217 cases of pulmonary neuroendocrine tumorfrom February 2012 to December 2018, were collected and sorted retrospectively. According to the following criteria, 179 cases were included in the study.
Case rejection criteria: When patient has both puncture and operation samples, the puncture samples are removed; The patient has only puncture specimen with less tissue, it is not suitable to be sectioned and stained again; The patient was pathologically diagnosed as mixed neuroendocrine tumor of the lung.
Immunohistochemistry and KRAS mutation test
In this study, the staining was conducted on tissue array. 4 um Sections were de-paraffinized and hydrated, and the endogenous peroxidase was blocked. Antigen retrieval was performed using the Dako Target Retrieval Solution, High PH (Dako Ominis, Agilent Technologies, Santa Clara, CA, USA), in a PTLink set at 98ºC for 25 min. Then slides were incubated with primary antibody for 60 min at room temperature. Immunostaining was achieved by an enzyme-conjugated polymer complex (Dako K8002, Agilent Technologies, Santa Clara, CA, USA) adapted for an autostainer (Dako Autostainer Link 48, Agilent Technologies). The primary antibodies used as follows: anti-Rb1 (Santa Cruz, sc−102, 1:250, USA), anti-P53 (Abcam, ab32389, 1:200, USA), anti-ASCL1 (Abcam, ab74065, 1:250, USA), anti-STK11 (Abcam, ab15095, 1:500, USA).
Interpretation standard of the markers above:
Under normal conditions, the half-life of p53 protein is very short and it is degraded quickly. The normal peripheral lung tissue was used as a wild-type control group, and immunohistochemical examination showed that the nuclei were scattered and brownish yellow. After mutation, the protein accumulated rapidly and stably located in the nucleus. Immunohistochemistry showed that more than 60% of tumor cells had uniform brownish yellow staining. In addition, TP53 gene can also produce nonsense mutation, and the expression of p53 protein in tumor cells is completely absent compared with the positive control in interstitial tissue. 60%−100% of the tumor cells were positive or negative (indicating mutation), the rest were wild type.
Rb1 encoded protein was located in the nucleus. In the normal cells, the nucleus is brownish yellow. After the gene mutation, the fibrovascular tissue was used as the positive control, and the expression was negative. Therefore, the expression of nuclear positive staining protein is normal, and the absence of nuclear staining suggests that Rb1 gene mutation.
STK11 gene encodes proteins that function in the cytoplasm. In adenocarcinoma, about 33% of STK11 can mutate, resulting in the loss of STK11 protein expression. Therefore, the lung adenocarcinoma was taken as the negative control, the tumor stroma as the positive control. The positive expression of STK11 protein indicated that STK11 was not mutated, and the deletion of STK11 protein indicated that STK11 gene mutation might exist.
As a nuclear transcription factor, ASCL1 protein is expressed in the nucleus, and the staining intensity of the nucleus is evaluated.
Immunohistochemical reactivity was scored by three pathologists (Zitian Huo, Yaqi Duan, and Guoping Wang) independently as follows: multi-plication of the intensity of immunostaining (1, weak;2, moderate; 3, strong) and the percentage of positivetumour cells, which resulted in a score of 0–300. Ascore < 10 was considered as 0 (cut-off level < 10), ascore of 10−40 was considered as 1+,41–140 as 2 + and 141– 300 as 3+.
KRAS gene detection kit (Amoydx company, China) was used as manufacture’s instruction.
Counting of mitosis in 2mm2 field of vision(count/HPF)
The number of mitotic images in the corresponding field of vision was calculated by using Nikon pathological microscope with the field number of eyepiece of 2. (actual field of view diameter = number of field of view / magnification of objective lens)
We obtained the survival data by consulting the original medical record, telephone and follow-up. Overall survival (OS) is defined as: from the day of disease diagnosis to the date of death or the last follow-up, calculated in months, the end date of follow-up is February 26, 2019, a total of 119 cases obtained OS information.
Integrate George Julies DNA/RNA sequencing data set and clinical prognosis data set,we totally obtained 57 case with Complete data information, including 14 cases of sclc-like type. Patients were divided into ASCL1-high/low group according to the mean fpkm.The survival analysis found that the different expression levels of ASCL1 cannot provide a good guide to the prognosis of patients in ovrall LCNEC levels. However, in sclc-like LCNEC, since there are only 14 patients with survival data, it can still be found that patients with high ASCL1 expression seem to have more Poor prognosis. Based on the statistics of our larger sample size, we can significantly find that patients with high ASCL1 expression in sclc-like LCNEC are more likely to have tumor metastasis and a worse prognosis.
The software graphpad prism 7.0 was used for statistical analysis. The overall survival (OS) was calculated by the Kaplan-Meier method and compared by the log-rank test. Two groups were compared using Student's t-test, and multiple groups were compared using one-way analysis of variance (ANOVA). Data were presented as the mean ± SEM. P < 0.05 was considered statistically significant.