| Part One Clinical and pathological characteristics of rectalneuroendocrine neoplasmsObjective: Clinicopathological characteristics of neuroendocrine neoplasm(NEN)are highly heterogeneous based on variable origins.Our study aims to explore the clinical features,guide the clinical diagnosis,treatment and assessment of rectal NEN.Methods: A total of 401 patients with histologically diagnosed gastroenteropancreatic neuroendocrine neoplasm(GEP-NEN)were retrospectively analyzed between July 2007 and July 2019.Sixty-two patients with rectal NEN were described in detail.The clinical manifestations,complete blood cell examination,endoscopic manifestations and histopathological characteristics of the patients were recorded and analyzed.Results:1.Incidence of rectal NEN was increasing year by yearA total of 401 patients with GEP-NEN in the Fourth Hospital of Hebei Medical University from July 2007 to July 2019 were collected in this study.Among them,there were 208 gastric NEN,23 small intestinal NEN,99 pancreatic NEN,9 colonic NEN,and 62 rectal NEN.The incidence of GEP-NEN was increasing year by year.Gastric NEN were the most common,followed by pancreatic NEN and rectal NEN,and small intestinal NEN and colonic NEN were rare.2.Clinical characteristics of rectal NENThere were 62 cases of rectal NEN,including 40 males and 22 females,with an average age of 54.5±12.0 years.There were significantly more males than females.There were 31 patients with the distance from the tumor to the anal verge ≤5cm,and 28 patients with the distance from the tumor to the anal verge >5cm.The main clinical manifestations were abdominal pain(18/62,29.0%)and changes in bowel habits(15/62,24.2%).The microscopic manifestations were mostly raised new organisms(51/62,82.3%).Distant metastases were found in 6 patients at the time of diagnosis,and the distant metastases were liver(5 cases)and bone metastases(1 case).Two patients developed distant metastasis during postoperative follow-up.Lymph node metastasis occurred in 21 cases,40 cases were without lymph node metastasis.The tumor size was recorded in detail in 58 patients,41 with tumors ≤ 1 cm,10 with tumors 1-2 cm,and 7 with tumors > 2 cm.Postoperative pathology of the rectum showed 44 cases of NETG1,10 cases of NETG2,6 cases of NEC,and 2 cases of Mi NEN.The depth of tumor invasion was 36 cases in T1,9cases in T2,7 cases in T3,and 10 cases in T4.5 patients died and 3 patients were lost to follow-up.The median survival time was not reached.The 1-year and 3-year survival rates were 94.1% and 82.5%,respectively.3.Comparison of neutrophil-to-lymphocyte ratio(NLR),Platelet-to-lymphocyte ratio(PLR)and Monocyte-to-lymphocyte ratio(MLR)between different clinical characteristics before treatmentAmong patients with rectal NEN,the mean NLR was 2.7±2.3,the mean PLR was 154.0±60.0,and the mean MLR was 0.26±0.20.There were statistically significant differences in NLR between groups of tumor grades(P<0.001),T stages(P=0.001),tumor sizes(P=0.026)and lymph node metastases(P<0.001).Patients with T3+T4,>1cm and positive lymph node metastases had a higher mean NLR.There were significant differences in PLR among age groups(P=0.003),and the mean PLR of patients ≤55 years was higher.There were statistically significant differences in MLR between groups with tumor grades(P<0.001),T stages(P<0.001),tumor sizes(P=0.017)and lymph node metastases(P<0.001).Patients with T3+T4,>1cm and positive lymph node metastases had a higher mean MLR.And the NLR value of dead patients was significantly higher than that of survivors(5.15 vs 2.60,P=0.002).Summary:The incidence of rectal NEN was increasing with the development of colorectal cancer screening programs.Most of the tumor size were smaller than 1 cm,and most of the rectal NEN were grade 1.Endoscopic tumor resection was the first treatment option.NLR and MLR values were higher in patients with NEC and Mi NEN,late tumor stage,larger tumor size,and positive lymph node metastasis.NLR may be a prognostic factor in patients with rectal NEN.Part TWO Metastatic risk score and prognostic factors of rectalneuroendocrine neoplasmObjective: To explore the factors affecting the prognosis,evaluate metastastic risk score for rectal NEN.To determine whether the distance of the tumor from the anal verge affects the patient’s prognosis.Methods: Clinical pathological and follow-up data of 62 patients identified as rectal NEN by pathology from July 2007 to July 2019 were carefully collected.Results:1.Patients’ symptomsAmong all rectal NEN patients,the primary clinical manifestation of17.7% patients was hematochezia.It was abdominal pain or discomfort in33.9% patients,it was alterations of bowel habits in 15(24.2%)patients,and in the remaining 15 patients(24.2%)had no clinical symptoms.2.Imaging featuresAmong 62 patients with rectal NEN,endoscopic performance of47(75.8%)cases showed submucosal bumps,5(8.1%)cases showed polypoid bumps,8(12.9%)cases showed ulcerative neoplasms,2 cases(3.2%)presented as nodules.The findings of abdominal enhanced CT of 27 cases were thickened.Five cases were found liver metastases and 1 case had a metastatic bone metastasis.3.Predictors of lymph node metastasisAmong 62 patients with rectal NEN,a total of 21 patients were in the presence of lymph node metastasis,40 patients had negative lymph nodes.Tumor sizes(P<0.001),tumor stages(P<0.001),and tumor grades(P<0.001)were associated with lymphatic metastasis.The multivariable exact logistic regression model showed that tumor stages(P=0.034)and tumor grades(P=0.001)were independent risk predictors of lymphatic metastases.We further developed a lymphatic metastasis risk score for 55 patients with rectal NEN and complete data to estimate lymph node metastasis risk.Patients with the score≥7.5 were more likely to develop lymph node metastasis(area: 0.958,95% CI 0.903-1.000,P<0.001).Sensitivity and specificity were 72.2% and97.3%).4.Risk factors associated with distant metastasisUnivariate analysis showed that tumor grade(P=0.002),depth of tumor invasion(P<0.001)and lymph node metastasis(P<0.001)were the factors affecting distant metastasis of rectal NEN.And multivariate analysis showed that tumor grade(P=0.016),depth of tumor invasion(P<0.001)and lymph node metastasis(P=0.001)were independent factors influencing on distant metastasis of rectal NEN.5.Survival of rectal NEN patientsAmong the 62 patients with rectal NEN,5 patients were dead,and 3 were lost to follow-up,with a follow-up rate of 95.2%.Median survival time of patients with NEC/Mi NEN was 26.0 months,while patients with NETG1 and NETG2 were not reached.There was none of the patients with a follow-up period of less than 6 months,8 patients between 6 months and 1 year,51 patients with more than 1 year,23 patients with more than 3 years,and 6patients with more than 5 years.Among the 51 patients who were followed up for more than 1 year,the 1-year survival rate of rectal NETG1 and NETG2 patients was 97.7%,and the 1-year survival rate of rectal NEC/Mi NEN patients was 75.0%.Among the 23 patients who were followed up for more than 3 years,the 3-year survival rate of rectal NETG1 and NETG2 patients was 94.4%,and the 3-year survival rate of rectal NEC/Mi NEN patients was40.0%.Among the 6 patients who were followed up for more than 5 years,2poorly-differentiated rectal NEC / Mi NEN died and 4 well-differentiated rectal NETG1 and NETG2 were still alive.Tumor stage(P=0.001),lymphatic metastases(P=0.012),tumor grade(P<0.001)and the distance from the tumor to the anal verge(P=0.022)were all associated with prognosis of patients.Patients with well-differentiated(NETG1 and NETG2)tumors,low T-stage(T1 and T2),without metastatic diseases,and lesions from the anal margin > 5 cm had a better prognosis.COX regression multivariate analysis showed that the tumor grade(P=0.004)and the distance from the tumor to the anal verge(P=0.012)were independent prognostic factors for patients with rectal NEN.Summary: Patients with tumor stage T3 and T4,NEC and Mi NEN were more likely to develop lymph node metastasis.Patients with well morphological differentiation(NETG1 and NETG2),low T-stage(T1 and T2)and without metastatic lymph nodes can have a relatively favorable prognosis.Patients with lesions from the anal margin > 5 cm and lymphatic metastasis risk score≥7.5 should be treated actively.Part THREE Prognostic molecular features and molecular types ofrectal neuroendocrine neoplasmObjective: Studies on genomic spectrum of rectal NEN are relatively few compared to other tumors.The gene mutations of rectal NEN are not very clear.We performed whole-genes NGS on tumor samples from 38 rectal NEN patients for mutation profiling and somatic copy number alteration(SCNA)analysis,aiming to identify genomic alterations associated with metastasis,distinct NEN subtypes to identify potential biomarkers associated with clinical outcomes.Methods:1.We retrospectively analyzed tumor samples from 62 rectal NEN patients treated at the Fourth Hospital of Hebei Medical University between2007 and 2019.Among these 62 tumor samples,38 specimens were performed whole-genome NGS,24 samples with small tumor volume were failure tested(Figure 1).Each sample was assessed for the Ki-67 index and differentiation(NET/NEC)based on independent pathologic evaluations by two pathologists.The detailed clinical information of all patients was included in Table 1.2.The relationship between metastatic / non-metastatic disease and clinicopathological characteristics was analyzed by chi-squared test.Fisher’s exact test was used to analyze relationship between differentially mutated genes and clinical characteristics.Gene mutation spectrum was drawn by R package.We performed mutation spectrum and mutation signature analyses using NMF.A KEGG database analysis was used to evaluate the pathway enrichment.Two-sided P<0.05 were considered significant.All analyses were performed in R 4.1.2.Results:1.Regional lymph node metastases and/or distant metastasis of rectal NEN were related to tumor sizes(P<0.001),tumor stages(P<0.001)and tumor grades(P<0.001).2.A total of 38 specimens were subjected to NGS,including 30 for NETG1,3 for NETG2,3 for NEC,and 2 for Mi NEN.The most recurrently altered genes were MUC16(21,55.3%),OBSCN(15,39.5%),FAT1(14,36.8%),ZFHX3(14,36.8%),AR(11,28.9%),and LRP2(11,28.9%).Different types of rectal NEN had differentially mutated genes.The results showed that the mutated frequency of APC,TP53,FPR1,FAT4,NOTCH2,BRCA1,FBXW7,NF1,SOX9,ANGPT2,KEL,NOTCH3,PDGFB and RPTOR genes in rectal NEC/Mi NEN was higher than rectal NETG1/G2.Although there was no statistical difference in these genes,such as FANCD2(5 cases),RUNX1(5 cases),TET3(5 cases),DAXX(6 cases),LTK(6 cases),MERTK(6 cases),PKN1(7 cases),FAT3(9 cases)and LRP2(11 cases),among different types of rectal NEN,however,these gene mutations were only found in G1 and G2 rectal NET.KMT2 C mutations were more common in rectal NET.BCL2L1,DAXX,LTK mutations occurred only in rectal NETG1.SPEN mutations occurred only in rectal NETG2.Mutations in the TP53,TYK2,APC,and PDGFB genes were common in metastatic rectal NEN.Patients with these mutated genes were at high risk of metastasis.3.Amplification of the oncogenes CALR(9 cases),DAXX(5 cases),BCL(5 cases),APEX1(3 cases),and NFKBIA(3 cases),and deletion of tumor suppressor genes CDKN2 A,CDKN2B,and MTAP were observed.There were no significant differences between gene amplification / deletion and clinicopathological features.4.The median tumor mutation burden(TMB)was 9.87(1.1-61.8)Mut/Mb,which was lower to that for the TCGA cohorts.The high TMB was observed in groups of age > 55 years(P=0.046),NEC and Mi NEN(P=0.040).TMB of NET G1 and NET G2 were significantly lower than that of NEC and Mi NEN(P=0.041).Older,NEC and Mi NEN patients may benefit from immunotherapy.5.Mismatch repair(P=0.030),Wnt(P=0.029),MAPK(P=0.016),Hedgehog(P=0.037)and PI3K/AKT(P=0.037)signaling pathways were involved in rectal NEN metastasis.Alterations in Wnt,MAPK,and PI3K/AKT signaling pathways promoted rectal NEN metastasis.The mismatch repair and Hedgehog signaling pathways were negatively correlated with rectal NEN metastasis.P53(P=0.009),Wnt(P=0.046)and TGFβ(P=0.019)signaling pathways were more pronounced in rectal NEC/Mi NEN.Alterations in these signaling pathways were associated with poorly differentiated NEN.6.C>T and T>C transitions were the most common forms of base substitution in rectal NEN.The mutation signatures were divided into eight types by NMF.Signature 3 and signature 8 were significantly represented as C>T / T>C base substitutions and were highly similar to the signatures described by SBS6 and SBS1 in the COSMIC database(similarities were 0.89 and 0.815,respectively).Signature 2 and signature 3 were correlated with DNA mismatch repair deficiency(SBS6).Signature 6 and signature 8 were correlated with spontaneous or enzymatic deamination of 5-methylcytosine(SBS1).Signature 1,signature 4 and signature 7 were associated with smoking(SBS5),Signature 5 was correlated with exposure to UV(SBS7a).Proportion of mutation signatures in each sample was analyzed.It was found that the main pathogenic cause of rectal NETG1 was DNA base modification and mismatch repair defects.Rectal NETG2,NEC and Mi NEN were mainly characterized by a mixture of signature 1,signature 2,signature 4,signature 5,signature 6 and signature 7.It was considered that rectal NETG2,NEC and Mi NEN were caused by a variety of factors including DNA mismatch repair defects,DNA base modifications,smoking and exposure to UV.7.DDR mutants were detected in 81.6%(31/38)of rectal NEN patients.The most significantly mutated genes were KMT2 C,MSH6,TP53 and FANCI.TMB of patients with DDR mutant genes was higher(P=0.029).It suggestted that patients with rectal NEN harboring DDR mutations may benefit from PARP inhibitors and immunotherapy.8.Rectal NEN were classified into three molecularly types by cluster analysis based on the mutant genes and signaling pathways combined with clinicopathological features.In type A,TP53,FANCA,NOTCH1,PLCG2,APC,PRKDC gene mutations were common,and the signaling pathways were Wnt,Notch,Cell.cycle and PI3K-AKT.Mostly were NETG2 and NEC with all tumor stages.8 patients had local / distant metastases.In type B,APEX1,DAXX,MSH6,SPTA1,CDH1,PKN1 gene mutations were common,and the signaling pathways were Apoptosis,Wnt,Notch.There was only one NETG2 and NEC,respectively,and the rest were all NETG1.Only one patient had tumor stage T3,and the rest were T1.Local / distant metastasis occurred in 2 patients.In type C,LRP2 gene mutation was more common,and MSH6 gene mutation was also being.The signal pathway was PI3K-AKT.All patients were NETG1.Only one patient had tumor stage T2,and the rest were T1.Only one patient had local / distant metastases.Type C was better than type A and type B in terms of tissue differentiation,tumor infiltration and metastases.It was found that LRP2 gene mutation was more common in rectal NET,and the PI3K-AKT signaling pathway was involved in the occurrence and development of rectal NET.Wnt and Notch signaling pathways were involved in the occurrence and development of high grade rectal NEN.Summary:In this study,rectal NEN were divided into three molecular types by clustering analysis of mutated genes and signal pathway changes combined with clinicopathological features.It was helpful for the evaluation of patient prognosis and the formulation of follow-up strategies.PARP inhibitors,MEK inhibitors and m TOR/AKT/PI3 K inhibitors may be effective drugs for the treatment of metastatic rectal NEN.Conclusion:1.Patients with rectal NEC / Mi NEN,advanced tumor stage,and larger tumor size had higher level of NLR and MLR and were more likely to develop lymph node metastases.Such patients need full evaluation and active treatment before treatment,and close follow-up after surgery.2.Pathological grade,T stage,lymph node metastasis,tumor distance from the anal verge and NLR are important factors affecting the prognosis of patients with rectal NEN.3.In this study,rectal NENs were divided into three molecular subtypes based on mutated genes and signaling pathways combined with clinicopathological features,and it was found that patients with LRP2 gene mutation had a better prognosis.PARP inhibitors,MEK inhibitors and m TOR/AKT/PI3 K inhibitors may be effective drugs for the treatment of metastatic rectal NEN. |
