The Role Of Telomere Prolonged Replacement Phenotype In The Progression Of Pancreatic Neuroendocrine Tumors And The Exploration Of Molecular Pathological Characteristics

Part 1:Exploration of the role of alternative lengthening of telomeres in pancreatic neuroendocrine neoplasms progression and its molecular pathological landscapeBackgroundPancreatic neuroendocrine neoplasms(PanNENs)are a heterogeneous group of lesions exhibiting different biological behavior.Some studies have already demonstrated the predictive valve of new WHO classification of PanNENs.However,the biological behavior of PanNENs vary even in the same stage or grade.The existing risk-assessment system,in a subset of PanNENs,do not accurately reflect the clinical behavior of these neoplasms.The alternative lengthening of telomeres(ALT)mechanism usually portends a poor prognosis of PanNENs according to the studies from Europe and the United States.The molecular genetic basis of ALT activation in sporadic PanNENs remains unclear,and the association of DNA damage repair protein defects and DNA methylation with ALT activation has not been studied.Our study of the prognostic significance of ALT activation in PanNENs,the molecular genetic background related to ALT activation,and the relationship between ALT activation and DNA damage repair pathways is reported here.Understanding ALT and how it is controlled by genetic and methylation pattern alterations will explain the heterogeneous behavior of ALT PanNENs,improve current prognosis assessment of PanNENs through the clinical,pathological and molecular scoring system and underpin the development of therapies for targeting neoplasms that depend on this mechanism for their continuing growth.Methods1.In a retrospective review of the surgical pathology files at Peking Union Medical College Hospital(PUMCH),we identified 156 patients with PanNENs who had samples available for studies.ATRX,DAXX,DNA repair proteins(MLH1,PMS2,MSH2,MSH6 and MGMT)expression were assessed by immunohistochemistry(IHC),ALT status were assessed by fluorescence in situ hybridization(FISH)and compared with clinicopathologic factors.2.The Methylation degree of 27 archival PanNENs specimens and 10 normal pancreatic tissues were investigated by Methylation specific-multiplex ligation-dependent probe amplification(MS-MLPA).Methylation differences were compared between ALT-positive and ALT-negative PanNENs specimens.3.Gene mutations in 30 pairs of PanNENs and corresponding normal tissues were detected by targeted next-generation sequencing and sanger sequencing.High frequency genes of germline mutations and somatic mutations were analyzed by biological information and statistics.The potential mutations associated with ALT and a poor prognosis in PanNENs were identified.Results1.The median length of follow-up was 53 months and ranged from 5 to 185 months.The estimated 5-,and 10-year survival rates were 91%,and 78%,respectively,higher than population-based studies in western.ALT-positive PanNETs displayed a significantly higher grade,Ki-67 index,size,and TNM stage(all,P<0.001).ALT also strongly correlated with lymph node metastases(P<0.001),distant metastases(P<0.001)and non-insulinoma(P<0.001).ALT-positive tumors were significantly associated with loss of ATRX/DAXX expression(P<0.001).Patients with ALT-positive PanNETs had a shorter recurrence-free survival than ALT-negative patients in G2 subgroup of PanNENs(P<0.001).In multivariate analysis,higher grade and stage were independent prognostic factors related to the risk of tumor progression in PanNENs.2.None determined loss of MLH1,PMS2,MSH2 and MSH6 were found in Pan NETs.Declined expression of MGMT was observed in 6.5%(9/138)patients,which was significantly associated with ALT(P<0.001).The probe MLH1 IV,MGMT ?showed increased methylation degree in ALT-positive vs.ALT-negative PanNENs(P<0.05).3.Nonsynonymous mutations in DAXX were present in 10%(3/30)of Pan NENs by sanger sequencing.MEN 1 was the most frequently and significantly mutated of these genes by targeted next-generation sequencing,present in 33%(10/30)of tumors.The mutations of DNA damage repair,chromatin remodeling-related genes and MEN1/DAXX/ATRX mainly converged in ALT positive tumors,only frequently mutations of MEN1/DAXX/ATRX affected samples were higher in ALT-positive than ALT-negative tumors(P=0.038).In addition,tumor suppressor genes,tyrosine kinase and receptor coding genes showed frequently mutations.ConclusionsThe estimated 5-,and 10-year survival rates of PanNENs were 91%,and 78%in our study,significantly higher than population-based studies in western.Patients with ALT-positive PanNETs had an increased risk of progression in G2 subgroup of PanNENs.Higher grade and stage were independent prognostic factors related to the risk of tumor progression in PanNENs.Loss of DNA mismatch repair protein expression and gene mutations are relatively low-frequency events in PanNENs.DNA methylation changes of MLH1 and MGMT were correlated with ALT activation.Mutations in DNA damage repair genes,chromatin remodeling genes and MEN1/DAXX/ATRX may be involved in ALT activation.Tumor suppressor genes,tyrosine kinases and their receptor coding genes play a role in the development of PanNENs.In conclusion,we confirmed that ALT-positive could be used to identify a subgroup with higher risk of progression in intermediate grade(G2)PanNETs.Changes in methylation degree of MLH1 and MGMT,somatic mutations in DNA damage repair,chromatin remodeling related genes and MEN1/DAXX/ATRX were likely to be required for ALT activation.Part 2:High minichromosome maintenance protein 7 proliferation indices:a powerful predictor of progression in pancreaticneuroendocrine neoplasms without distant metastasis at the time of surgeryPancreatic neuroendocrine neoplasms(PanNENs)have an unpredictable clinical course that varies from indolent to highly malignant.No immunohistochemical markers are available for reliable prediction of the biological behavior of early-stage PanNENs.Minichromosome maintenance protein 7(MCM7)is a putative powerful marker of cell proliferation.Whether the expression of MCM7 is related to the risk of PanNENs progression remains unclear.We assessed the clinical behavior of 156 PanNENs with respect to stage,grade,Ki-67 index,MCM7 index,and other pathologic features.A high MCM7 index was significantly associated with larger tumor size(P<0.001),nonfunctioning tumor(P<0.001),increased grade(P<0.0001),and later TNM stage(P<0.001).In multivariate analysis,G2/G3(hazard ratio(HR),2.21;95%confidence interval(CI),1.35-3.62;P<0.001),stage ?/?(HR,2.11;95%CI,1.31-3.41;P<0.001),and MCM7 labeling index(LI)>5%(HR,3.81;95%CI,1.30-11.17;P=0.02)were independent negative prognostic factors related to the risk of tumor progression in stage ?-? disease.MCM7 LI>5%was associated with an increased risk of progression in stages?/?,?-?,and ?-?.Our study confirms that MCM7 is a valuable marker for assessing the progression of PanNENs,especially in patients with early-stage disease and without distant metastasis.