Molecular Mechanism Of Primary Progression To High-grade Tumors In Gastrointestinal Stromal Tumors

?Background? Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors in the digestive tract,with the different biological behaviors and clinical manifestations ranged from benign to malignant.The risk stratification of primary GIST relies on the tumor location,tumor size,mitotic index and tumor rupture.The different molecular characteristics between benign and malignant GIST are still unclear.Most malignant GISTs are characterized by uniform high-grade sarcomatoid histology in multiple nodules or single nodule,but a few cases show co-existing low-grade tumors in the same nodule or partial nodules.It is still controversial that malignant GIST is arose form the initial onset,or from the progression of low-grade tumor.Recently,genomic muation analysis and protein expression study,basing on individual patients,showed that the high-risk GISTs have different gene mutations and protein expression compared with low-grade one,but it is not reported on the molecular mechanism of tumor primary progression from low-grade GIST to high-grade one basing on a series of cases with concomitant both components.?Objectives? This study is to investigate the differences in morphological characteristics and immunohistochemical expression of the paired high-grade and low-grade GISTs.Whole-exome sequencing is used to detect differential mutations between high-grade GISTs and low-grade tumors,and the possible functions of mutant genes are further analyzed and confirmed.This study may provide a new basis for the accurate prognosis stratification of GIST.It is expected to explain the malignant progression mechanism of some GISTs,and to provide some new molecular markers for predicting prognosis and guiding treatment.The purposes of this research mainly include:1.To explore the morphological differences and immunohistochemical expression differences between the high-grade tumor and low-grade one co-occurred in the same patient.2.To analyze the differential genetic mutations in high-grade GIST components compared with low-grade one,and to explore the possible molecular mechanisms that the genetic mutations promote primary progression to high-grade GISTs.3.To analyze the MYC copy number variation(CNV)basing on whole-exome sequencing data,and to detect the MYC CNV in tissue samples of GIST,and to investigate the molecular mechanisms of MYC CNV acting in progression of GIST.4.To detect the function of MGA in GIST cell proliferation,and to explore its related molecular mechanisms on inhibition of proliferation.5.To investigate the expression of Cyclin D1 and p16INK4 A in GIST,and to explore their molecular functions in tumor progression.?Methods? The histopathological observation,immunohistochemistry,high-throughput sequencing,fluorescence in situ hybridization(FISH)and molecular biology experiments were used in this study,and the main examples are as follows:1.The clinicopathological features of 22 cases of GIST with concomitant low-and high-grade tumors were retrospectively analyzed,and the prognosis of these patients was followed up.2.Whole-exome sequencing(WES)of 10 pairs of matched high-grade and low-grade GIST samples was carried out,and basic information analysis and advanced bioinformatics analysis were performed to compare the differential gene mutations and CNV between high-grade GIST and low-grade tumor.3.Sanger sequencing was employed to verify the differential tumor-associated gene mutations;and FISH was performed to verify and detect MYC copy number gain inGIST.4.Western Blot was used to detect the protein expression of MGA,MAX,MYC,p16INK4 A and Cyclin D1 in GIST cell lines.5.A GIST cell line with low expression of MGA was constructed by using lentivirus-mediated sh RNA interference targeting GIST-T1.6.Cell growth curve and plate cloning experiments were used to detect the proliferative activity of GIST cell lines.7.Immunohistochemical staining was carried out to detect the expression of Cyclin D1 and p16INK4 A in GIST tissue samples.?Results?1.All patients with coexisting high-and low-grade GIST did not receive neoadjuvant therapy with imatinib prior to surgery.The tumors mostly occurred in the small intestine(16 cases)and in the stomach(5 cases),with the diameter of not less than 4 cm.Nine cases showed multifocal lesions,and six cases occurred metastasis at the time of diagnosis.One patient presented with postoperative recurrence half a year after surgery,and 4 cases died within 4 years after surgery.Low-grade tumors showed a lower cell density and spindle cells with low mitotic index(1~2/50 high power fields(HPF));high-grade tumor revealed a higher cell density and short fusiform or epithelial cells with obvious atypia,and mitotic figures are more common(6~90/50 HPF).The expression of CD34 was significantly decreased in the high-grade tumors compared with low-grade tumors,and Ki-67 proliferation index of these two components was significantly different.2.Compared to the human reference genome GRCh37/hg19,802 mutations in 247 tumor-associated genes were detected in 20 cases of GIST samples by WES.193(78.14%)genes mutated simultaneously in high-grade GIST and low-grade components,and 30(12.15%)genes only in high-grade tumors,and 24(9.71%)genes only in low-grade tumors.3.Basing on WES data,all 10 patients revealed the same KIT mutations in both high-grade GISTs and matched low-grade tumors.4.35 mutations in 30 tumor-associated genes,that occurred only in the high-grade components of GIST,were detected by WES,and nine genes mutated only in high-grade tumors were confirmed by Sanger sequencing,namely MGA,SDHA,ARID1A,LATS2,MAX,SETD2,RB1,RPS6KB2,and PIK3 CA.MGA mutations were detected in 2 cases of small intestinal GIST.KEGG pathway analysis suggests that these differential tumor-associated genes are mainly involved in cell cycle regulation,apoptosis inhibition,and chromosome remodeling.5.Compared to the c Bio Portal database,seven genes are considered to be recurrent mutant genes,namely RB1,MAX,SETD2,SDHA,ARID1 A,MGA and PIK3 CA.Except for the last one,all genes are the top 20 common mutant genes in GIST.The mutations of LATS2 and RPS6KB2 were reported for the first time in GIST.6.Copy number gain of MYC gene was detected in 3 cases of high-grade GIST compared to matched low-grade component by WES.MYC copy number amplification was confirmed by FISH in one case of high-grade tumor.The other two cases showed extra copy of chromosome 8 with copy number gain of MYC gene.7.In MGA sh RNA lentivirus-infected GIST cell line,the expression of p16INK4 A was lower than that in normal and negative control cell lines,while the expression of Cyclin D1 was increased.Furthermore,MGA sh RNA lentivirus-infected GIST cells had higher proliferative activity and higher colony forming ability.8.High-grade GIST samples revealed lower immunohistochemical expression for p16INK4 A,and its expression was negatively correlated with mitotic index,Ki-67 proliferation index,risk stratification and tumor maximum diameter.Cyclin D1 was highly expressed in high-grade tumors,and its expression was positively correlated with mitotic index and tumor maximum diameter.9.Among the 130 GIST samples,50.77% of the samples had MYC copy number gain,and all of them showed a low increase of copy number.In GIST with concomitant low-and high-grade tumors,the high-grade component revealed more MYC copy number gain than matched low-grade tumor.In pure low-grade or high-grade GIST,high-grade tumors had a higher incidence of MYC copy number gain than low-grade tumors.The incidence of MYC copy number gain in low-grade GISTs coexisted with high-grade tumors was significantly higher than that in pure low-grade cases.Moreover,copy number gain of MYC gene was positively correlated with mitotic index and Ki-67 proliferation index,and negatively correlated with p16INK4 A expression in pure low-grade or high-grade GISTs.?Conclusions? 1.GISTs with coexisting high-and low-grade tumor components often occurred in the small intestine.Tumor metastasis was common,and patients often had a poor prognosis.High-grade tumors usually revealed short fusiform or epithelial cells with obvious atypia,high mitotic index and Ki-67 proliferation index,and necrosis was often occurred.CD34 expression in high-grade component was decreased than that in low-grade one.2.Whole-exome sequencing data showed that the high-grade tumors and matched low-grade components had most of the same genetic mutations,including KIT mutations,suggesting that both may originate from common progenitor cells.As the tumor grows,different mutations occurred in some components,which may lead to the formation of tumor heterogeneity and the malignant differentiation of tumor.3.Differential mutations that occurred only in high-grade GISTs might be primary molecular events in tumor progression,which may be the result of independent or synergistic effects of multiple genes through different cell signaling pathways.In particular,mutations in cell cycle regulation-associated genes may promote tumor cell proliferation by regulating cell cycle,leading to GIST progression from low-grade tumor to high-grade tumor.4.The MGA/MAX/MYC network might play an important role in GIST tumor progression.Reduced expression of MGA promoted GIST cell proliferation and cell clony formation,possibly through the p16INK4A/CDK4/Cyclin D1 cell cycle regulatory pathway.5.MYC copy number gain in GIST was a common genetic change,which may occur in the early stage of tumor,and low-grade GIST accompanied by MYC copy number gain were more likely to progress to higher grade tumor.GIST with MYC copy number gain had higher proliferative activity,and was negatively correlated with expression of p16INK4 A.6.Reduced expression of p16INK4 A and increased expression of Cyclin D1 might promote cell proliferation,resulting in GIST progression to a high-grade tumor.In conclusion,high-grade GISTs had different morphological characteristics and immunohistochemical expression compared to coexisting low-grade tumors.The primary progression of GISTs might be the result of a variety of different genes acting throughdifferent signaling pathways,in which,cell cycle regulation-related gene mutations,copy number gain of MYC gene and dysregulation of cell cycle regulator might play important roles.