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FREQUENCY OF LOSS OF SUCCINATE DEHYDROGENASE-B IN GASTROINTESTINAL STROMAL TUMORS: SHAUKAT KHANUM HOSPITAL EXPERIENCE.

Byline: Syed Saad Gardezi, Asif Loya, Noreen Akhter, Sajid Mushtaq, Usman Hassan, Zain Mehdi and Omer Waqas

ABSTRACT

Objective: To assess the frequency of loss of expression of succinate dehydrogenase-B (SDH-B) in gastrointestinal stromal tumors (GISTS) of our population through immunohistochemistry.

Study Design: Descriptive study.

Place and Duration of Study: Department of Pathology, Shaukat Khanum Hospital and Research Centre, Lahore for a duration of 6 months, Apr 2015 to Sep 2015.

Patients and Methods: A total of 63 diagnosed cases of GISTS suitable by inclusion criteria were included in the study. The blocks of already diagnosed cases was reviewed by 2 pathologists and after confirmation SDH-B immunohistochemical stain was applied with normal tissue as its control. Lack of staining in tumor was interpreted as "Loss of expression".

Results: Mean age of our population was n=44.83 +- 17.76 years. The most common tumor site was small intestine n=23 (63.5%) cases, followed by stomach, n=15 cases. CD117 was positive in all the tumors. Only n=3 tumors were found to be SDH-B deficient. All of these tumors involved the stomach and had a median age of 20 years. All of these tumors had an epithelioid morphology.

Conclusion: Immunohistochemistry was found reliable and cost-effective method for detecting mutation in SDH gene. Based on findings of this study it is recommended that all gastric GISTs below the age of 30 years with epithelioid morphology should be tested for SDH deficiency.

Keywords: Gastrointestinal stromal tumors, Immunohistochemistry, SDH-B.

INTRODUCTION

Gastrointestinal stromal tumors (GIST) is the most common mesenchymal tumor of the gastrointestinal tract comprising 11-19.6 cases per million. The adult form of GIST mainly presents in the sixth decade of life and shows no gender predilection. Although, about one fifth of the tumors are asymptomatic, other tumors present with abdominal pain, gastrointestinal bleeding and symptoms of obstruction1. Stomach is the most common location followed by small bowel. It is mainly a submucosal and mural lesion. Surgical excision is the treatment of choice for most tumors2. In unresectable and metastatic cases targeted drug Imatinib is used, which is a tyrosine kinase inhibitor (TKI)3. Most common type of molecular alteration in GIST is c-Kit mutation followed by platelet derived growth factor receptor (PDGFR) mutations which make up a combined 95% of the tumors.

The rest of the 5% of the tumors were previously thought to be wild type tumors, but now they include tumors with mutations in succinate dehydrogenase-B (SDH-B) and BRAF. SDH-B deficient tumors include Pediatric type GISTS and those associated with Carney Stratakis syndrome4. Succinate dehydrogenase consists of four protein subunits: SDHA to SDHD that are localized in the inner mitochondrial membrane and act at the interphase of the tricarboxylic acid cycle and electron transport chain. Loss of function of SDHB, SDHC or SDHD is central to the pathogenesis of these tumors. These tumors comprise 7.5% of all gastrointestinal stromal tumors and include a group of children, young adults and a small number of older individuals. This variant only occurs in the stomach and are commonly multi-focal with predominant epithelioid morphology5.

Deficiency of succinate dehydrogenase can be detected by either molecular methods or by immunohistochemistry. Molecular methods involve laser microdissection and Sanger sequencing. Antibody to succinate dehydro-genase, especially to SDH-B, has shown good correlation with molecular analysis5.

Normal tissue shows granular cytoplasmic staining of SDHB reflecting the presence of this enzyme in the mitochondria. The SDH deficient cases show loss of expression and this correlates with mutation in genes encoding this protein. The purpose of this study is to assess the frequency of SDHB deficient GISTS in our cases through immunohistochemistry. These tumors show poor response to Imatinib and behave aggressively with propensity for distant and nodal metastases. Therefore, identification of this type of GIST prompts appropriate measures for management.

MATERIAL AND METHODS

This was an observational study (descriptive study) carried out in the department of Pathology, Shaukat Khanum Hospital and Research centre, Lahore, Pakistan from April 2015 to September 2015. Total number of 63 diagnosed cases of GIST were included through purposive non-probability sampling technique throughout the years 2011 to 2014. Both excison and incision biopsies were included. Reports and slides of all these cases were retrieved. The sample population included patients of all ages, gender, tumor size, tumor site and histological subtypes of GIST. Our exclusion criteria included unfixed or necrotic tissue, history of previous chemotherapy or recurrent tumors.

The slides were reviewed for diagnosis by two histopathologists with special interest in GI pathology. Parameters including histological subtype, number of mitosis per 20/HPF and risk assessment were reviewed. Immuno-histochemical expression of diagnostic markers of GIST CD34, CD117 and DOG-1 were reassessed and documented. SDHB (concentrated rabbit monoclonal antibody, clone EP288 of abcam) immunohistochemical stain was applied to all the blocks with a dilution of 1:20. Non-neoplastic tissue in the blocks was used as internal positive control. Lack of staining in tumor was interpreted as "Loss of expression". The analysis was done through SPSS 22.0. Mean +- S.D was calculated for quantitative variables like age, greatest tumor dimension (T) and mitosis. Frequencies assessed for qualitative variables like gender, risk assessment, and expression of Immunohisto-chemical markers including SDHB.

Table-I: Clinico-pathological characteristics of patients included in the study.

Site###No. of cases###Gender###Histological subtypes###Mean size(cm)**###Mean no of mitosis(per 20HPF)###Risk stratification**###Immuno-histochemical makers Positivity(%)

###Male###Spindle###Epitheloid###Mixed###Low###Moderate###High###CD117###DOG 1###CD 34###SDHB*

###Female###cell###type###Risk###Risk###Risk###(n=63)###(n=19)###(n=11)###(n=63)

Stomach###15###9(60%)###12(80%)###3(15%)###0###8.9 +- 5.1###7.1 +- 9.3###6###2###4###100%###80%###50%###20%

###6(40%)

Small bowel###23###15(65.2%)###19(82.6%)###1(4.3%)###3(13.04%)###9.8 +- 2.9###6.1 +- 5.2###1###2###13###100%###100%###75%###0%

###8(34.8%)

Large bowel###4###3(75%)###4(100%)###0###0###11.7 +- 3.4###29.8 +- 26.7###0###0###4###100%###100%###-###0%

###1(25%)

Abdomen(NOS)###21###13(61.9%)###18(85.7%)###2(9.5%)###1(4.7%)###13.7 +- 5.9###15.4 +- 16.6###1###0###10###100%###90%###50%###0%

###8(38.1%)

Table-II: Clinicopathological features of SDH-B deficient GISTS.

Mean age###22.67 +- 6.4 years

Median age###20 years

Mean size###8.0 +- 6.4 cm

Spindle cell type###0

Epithelioid cell type###3

Site###Stomach(3 out of 3)

Risk assessment###Low risk(n=1), Moderate risk(n=2),

###High risk(n=0)

Table-III: Important clinical and pathological features of SDHB deficient cases in study.

Case no.###Age###Gender###Site###Type of Biopsy###Histological type###Size(cm)###Mitosis###Risk stratification###Margins

1###20###Male###Stomach (Not###Excision###Epithelioid###15###4###Moderate Risk###Not

###specified)###(Fragmented)###assessable

2###30###Male###Antrum###Excision###Epithelioid###3.5###8###Moderate Risk###Negative

3###18###Female###Lesser###Excision###Epithelioid###5.5###4###Low risk###Negative

###curvature

RESULTS

Mean age of the population was n=44.83 +- 17.76 years ranging from 17 years to 90 years. There were n=40 (63.5%) males and n=23 (36.5%) females. There were n=40 excision biopsies and n=23 incision biopsies. The most common site of tumor in our study was small intestine n=23 (36.5%) cases followed by stomach, n=15 (23.8%) cases and large bowel, n=4 (6.3%) cases and n=21 cases (33.33%) were extra gastrointestinal. Mean size of tumor was 10.74 +- 4.9 cm. Most tumors were spindle cell type n=53 (84.1%) followed by pure epithelioid type n=5 (7.9%) and mixed spindle and epithelioid type, n=4 (6.3%). The mean number of mitoses was 11.13 +- 14.13 per 20/HPF. Risk stratification was only carried out in the excision biopsies (n=40) or in few cases of incision biopsies (n=3) where radiological information was available. Majority of the tumors were included in the high risk category (n=31, 72%). N=4 cases (9.3%) belonged to intermediate type and n=8 (18.6%) cases represented the low risk category.

Breakdown of clinico-pathological features is presented in table-I. All the tumors (n=63) were positive for CD 117. DOG 1 was performed in n=19 cases, of which 17 were positive. While, CD34 was performed on only n=11 of the cases, and seven of them were positive. There was loss of expression of SDHB in n=3 cases (4.7%). Clinicopathological characteristics of SDHB deficient GISTs are summarized in table-II, clinical and pathological features of SDHB ar summarized in table-III.

DISCUSSION

GISTS are the most common mesenchymal tumors of the gastrointestinal tract1. GISTs occur throughout the tubular gut as well as in omentum, mesentry, pelvis and retroperitoneum. GIST usually present as solitary, rounded lobular masses in the muscularis propria or the submucosa (fig-1). They have a firm gray white cut surface. Microscopically, GIST can show either spindle cell morphology or epithelioid cell morphology. The spindle cell type which comprises of the majority of tumors has uniform pale eosinophilic cytoplasm, oval to short spindled nuclei, paranuclear vacuoles and skeinoid fibers (fig-2). About third of the tumors have epithelioid morphology with polygonal cells, round to oval nuclei and cytoplasmic retraction2. About 7-10% of GISTs have loss of function of the succinate dehydrogenase complex on inner mitochondrial membrane. SDHB deficiency is identified in many tumors including renal tumors, paragangliomas and pheochromocytomas.

The current study had a sample population of n=63 cases. The median age of our population was 40 years which is far less from the classic study by Miettinen et al, who reported a median age of 63 years6, but closer to another Pakistani study that reported a mean age of 51 years11. Both these studies showed male predominance similar to the current study. Spindle cell histology was also the most frequent type in the study by Din et al. The same study also showed a large number of high risk cases in stomach and small intestine7.

About n=15 (23.8%) cases were gastric in origin in our study. A fifth of these cases were negative for SDHB. This number is far higher than a previous study by Miettinen et al, who found SDHB mutations in only 7.5% of all the gastric GISTS he examined. However, it was a larger study with more than 700 cases of gastric GISTS6. Although, the current study might still imply that SDHB deficient GISTS are more prevalent in our society, a larger study is recommended to confirm this finding5. All of the cases in our study were under the age of 30 years which is younger than the average age of non-SDH deficient cases in our study. The median age of SDHB deficient tumors in the current study was 20 years. A similar trend was also reported by Miettinen et al, who reported a median age of 21 years in their study5. Most studies reported a female predominance in their study, but in our study there was male predominance as a whole.

All of SDH deficient patients showed epithelioid morphology in our study. In contrast, only 7.9% of non SDH deficient GISTS showed pure epithelioid features. The predominance of epithelioid phenotype is also apparent in most of the larger studies. No significant difference was found between tumor size, mitosis and risk category of SDH deficient and intact tumors. Most SDH deficient tumors are known to show prognosis independent of all these factors. CD117 and DOG1 were positive in all SDHB deficient GISTS. The Pediatric type GISTs (SDH-B negative) have been reported to have a more indolent course than adult GIST, and most studies have reported a female predominance. The tumor is frequently multi-focal and shows frequent recurrence. There is an increased incidence of nodal and visceral metastasis, including liver and abdominal sites. The tumor responds poorly to the conventional therapies and therefore identification of this tumor is of paramount importance8-9.

This is the first study on the expression of SDHB in cases of GISTs in Pakistan and we hope that expression of SDHB will become an integral part of diagnostic and treatment protocol for management of GIST.

CONCLUSION

Immunohistochemistry is a reliable and cost-effective method for detecting mutation in SDH gene. Based on findings of this study it is recommended that all gastric GISTs below the age of 30 years with epithelioid morphology should be tested for SDH deficiency.

CONFLICT OF INTEREST

This study has no conflict of interest to declare by any author.

REFERENCES

1. Soreide K, Sandvik OM, Soreide JA, Giljaca V, Jureckova A, Bulusu VR. Global epidemiology of gastrointestinal stromal tumours (GIST): A systematic review of population-based cohort studies. Cancer epidemiology 2016; 40: 39-46.

2. Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and prognosis at different sites. In Seminars in diagnostic pathology 2006; 23(2): 70-83.

3. West RB, Corless CL, Chen X, Rubin BP, Subramanian S. The novel marker, DOG1, is expressed ubiquitously in gastro-intestinal stromal tumors irrespective of KIT or PDGFRA mutation status. Am J Pathol 2004; 165(1): 107-13.

4. Janeway KA, Kim SY, Lodish M, Nose V, Rustin P, Gaal J, et al. Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations. Proc Natl Acad Sci USA 2011; 108(1): 314-8.

5. Miettinen M, Killian JK, Wang ZF, Lasota J, Lau C, Jones L, et al. Immunohistochemical loss of succinate dehydroge-nase subunit A (SDHA) in gastrointestinal stromal tumors (GISTs) signals SDHA germline mutation. Am J pathol 2013; 37(2): 234.

6. Miettinen M, Sobin LH, Lasota J. Gastrointestinal stromal tumors of the stomach: A clinicopathologic, immunohisto-chemical, and molecular genetic study of 1765 cases with longterm follow-up. Am J Surg Pathol 2005; 29(1): 52-68.

7. NU Din, Ahmad Z, Arshad H, Idrees R, Kayani N. Gastro-intestinal stromal tumors: A clinicopathologic and risk stratification study of 255 cases from Pakistan and review of literature. Asian Pac J Cancer Prev 2015; 16(12): 4873-80.

8. Miettinen M, Furlong M, Sarlomo-Rikala M, Burke A, Sobin LH, Lasota J. Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the rectum and anus: A clinicopathologic, immunohistochemical, and molecular genetic study of 144 cases. Am J Surg Pathol 2001; 25(9): 1121-33.

9. Miettinen M, Lasota J. Gastrointestinal stromal tumors: Pathology and prognosis at different sites. Semin Diagn Pathol 2006; 23(2): 70-83.
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Publication:Pakistan Armed Forces Medical Journal
Geographic Code:9PAKI
Date:Aug 31, 2018
Words:2557
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