Immunohistochemical Phenotype Of Pulmonary Neuroendocrine Tumors And Its Prognostic Implication

Background:Pulmonary neuroendocrine tumors are a group of heterogeneous tumors that originate from bronchial Kulchitsky neuroendocrine cells.Based on the cell morphology,degree of necrosis,and cell proliferation status,pulmonary neuroendocrine tumors could be divided into four subtypes:typical carcinoid?TC?,atypical carcinoid?AC?,large cell neuroendocrine carcinoma?LCNEC?,and Small cell lung carcinoma?SCLC?.TC and AC belong to well-differentiated neuroendocrine tumor?WD-NET?while LCNEC and SCLC belong to poorly-differentiated neuroendocrine cancer?PD-NEC?.The genome and transcriptome of pulmonary neuroendocrine tumors showed that WD-NET and PD-NEC have significantly different molecular genetic changes.Therefore,accurate diagnosis of pulmonary neuroendocrine tumors is extremely important.However,in clinical work,we often encounter cases that are difficult to classify according to diagnostic criteria,and the classification is not refined in the diagnosis.Objective:This study aimed to evaluate the expression levels of NEC-related molecular markers in different neuroendocrine tumors through immunohistochemistry,and further to investigate the molecular typing of LCNEC.Besides,we investigated whether WD-NET G3 cases existed in lung tumors and assessed the prognostic value of clinicopathological features in different phenotype.Methods:According to cell differentiation?including morphology and necrosis?,mitotic count,and Ki67,179 cases of neuroendocrine tumors in the pathology department of Tongji Hospital from 2012 to 2018 were re-examined by three pathologists.IHC was used to detect the different expression of molecular prototypic related proteins p53,Rb1,Ascl1 and Stk11.RT-PCR was used to detect the gene mutation of KRAS.The clinical pathology and prognosis information of the patients were collected to evaluate the prognostic value of different subtypes among LCNEC.Results:1.By morphological analysis of 179 cases of pulmonary neuroendocrine tumors,pathologists agreed that 29 cases of TC/AC,88 cases of SCLC met all diagnostic criteria;43 cases LCNEC strictly met the diagnostic criteria while Ki67>60%;There were 19 cases which did not meet the diagnose criteria,and they were classified as undefined temporarily.Among those cases,3 cases had a carcinoid morphology,with Ki67>20%or mitotic count>10/2mm²;The morphology of the remaining 16 cases was between WD-NET and PD-NEC,of which Ki67 was between 20%and 60%.2.The expression of p53,Rb1,Ascl1,and Stk11 in neuroendocrine tumors was evaluated.The protein expression of p53 and Rb1 was significantly different between WD-NET and PD-NEC.In 29 cases of WD-NET,the expression of p53 protein was absent in only one case of AC while the expression of Rb1 was all in right status.The PD-NECs shared a common p53 mutant expression pattern(p53^+/-)and a loss expression of Rb1.In SCLC,p53mutant expression and Rb1 deletion were found in 93%?82/88?and 92%?81/88?cases.In LCNEC,the indicators above were 91%?39/43?and 77%?33/43?,respectively.The abnormal expression ratios of Stk11 protein in WD-NET,SCLC and LCNEC were 21%?6/29?,0%?0/88?and 9%?4/43?,respectively.Compared with WD-NET,the high expression ratios of Ascl1 were 31%?9/29?,76%?33/43?and 76%?67/88?in WD-NET,LCNEC and SCLC,respectively.3.Molecular typing of SCLC,LCNEC,and unclassified cases was classified by immunohistochemical protein expression and KRAS gene mutation detection.1).According to the expression level of Ascl1,SCLC can be divided into classic SCLC(SCLC Ascl1^high)and SCLC Ascl1^low.2).In 43 typical LCNEC,29 patients had both p53 and Rb1 protein abnormal expression(p53^wt+Rb1^-),which can be classified into SCLC-Like LCNEC.Among them,16 cases were SCLC-Like Ascl1^high subtype;13 cases were SCLC-LikeAscl1^low subtype?also known as LCNEC-Type II?.1 case of p53^wtRb1^-Ascl1^lowow with KRAS^mut and 4 cases of p53^+/-Rb1⁺Ascl1^high/low with Stk11^-/KRAS^wt were classified into NSCLC-Like LCNEC,and 1 case belonged to p53^+/-Rb1⁺Ascl1^high?LCNEC-Type I?.The other 3 cases belonged to p53^wt Rb1⁺Ascl1^high/low and 5 cases belonged to p53^+/-Rb1⁺Ascl1^high/low,which had no Stk11 expression loss and KRAS gene mutation,and could not be classified into the existing literature,namely LCNEC-undefined.There were 19cases of NEC that were not classified.8 cases of p53^+/-Rb1^-can belong to SCLC-Like LCNEC,among them 3 cases were Ascl1^high subtype and 5 cases were Ascl1^low subtype.Only 4 cases showed abnormal expression of p53,without Rb1,Stk11 deletion or KRAS mutation,which is difficult to classify at present;7 cases are p53^wtRb1⁺cases with high proliferative activity,considered as WD-NET G3.4.Through prognostic survival analysis of different types of neuroendocrine tumors,the survival tendency of high-proliferation p53^wtRb1⁺type was between WD-NET and PD-NEC.There was no significant difference in survival rate between SCLC and LCNEC;In SCLC,there was no significant difference in survival between Ascl1^high or Ascl1^low subgroups;And there was also no significant difference between SCLC-Like and NSCLC-Like+LCNEC-undefined in LCNEC.But the SCLC-like Ascl1^highigh subgroup had a statistically significant higher lymph node metastasis rate and worse prognosis than the SCLC-like Ascl1^low subgroup.In LCNEC,the expression level of Ascl1 is only related to lymph node metastasis,and there is no difference in survival.Conclusion:1.The expression of p53,Rb1,Stk11 and Ascl1 in different neuroendocrine tumors is significantly different.By immunohistochemical combination of four proteins?especially combined with p53 and Rb1?and KRAS mutation gene detection,some can be classified as high-proliferation of p53^wtRb1^-cases,which suggests the existence of WD-NET G3.And can help us to carry out preliminary molecular typing on SCLC and LCNEC.2.The prognostic analysis after immunohistochemical molecular typing indicated that the survival rate of high-proliferation p53^wtRb1^-type was between WD-NET and PD-NEC.In LCNEC,lymph node metastasis was more likely to occur in SCLC-Like Ascl1^high subgroup than the SCLC-Like Ascl1^low subgroup,and the prognosis in Ascl1^high sub-population is worse.Immunohistochemical molecular typing is helpful for clinical evaluation of patient prognosis.