Clinical Features And Prognosis of Advanced Intra-And Extra-Pulmonary Neuroendocrine Carcinomas

Objective: We analyzed the clinical features and prognosis of advanced intra- and extra-pulmonary neuroendocrine carcinomas (NECs) in order to provide further guidance for the clinical treatment of small-cell lung cancer, which is a type of advanced intrapulmonary NECs. Methods: The clinical data and survival of 123 patients with advanced intra- and extra-pulmonary NECs in the Fujian Medical University Union Hospital, Fujian Province, China, between January 2013 to November 2019 were collected. We retrospectively analyzed the corresponding clinical diagnosis and treatment, and explored the relevant factors affecting the survival prognosis of patients with intra- and extra-pulmonary NECs. Results: The data of 123 patients were collected. There were 90 cases of intra-pulmonary NECS (including 81 cases of small-cell lung cancer, SCLC), 25 cases of extra-pulmonary NECs involving in the gastrointestinal tract, and 8 cases of extra-pulmonary NECs in other regions. The median overall survival (OS) of intra-pulmonary NECs was 13.53 months, of which the median OS of SCLC was 12.97 months, and the median OS of other intra-pulmonary NECs was 27.07 months. The median OS of extra-pulmonary NECs in the gastrointestinal tract was 9.42 months, and the OS of extra-pulmonary NECs in the other regions was 8.69 months. The median OS of intra-pulmonary NECs was signicantly longer than that of the extra-pulmonary NECs in the gastrointestinal tract and in the other regions (P < 0.05). Multivariate analysis showed that age, liver metastasis, number of cycles of rst-line chemotherapy, and chest radiotherapy were risk factors affecting OS in patients with NECs (P < 0.05). Conclusions: The survival of intra-pulmonary NECs was signicantly longer than that of extra-pulmonary NECs of the gastrointestinal tract and in other regions. However, patients with advanced intra- and extrapulmonary NECs who were older and had liver metastases had a poorer prognosis. Multi-disciplinary treatments such as multi-cycle chemotherapy and combination of chemotherapy and radiotherapy should play an important role in prolonging the survival of NECs.

The incidence of NENs was previously considered to be low. However, the number of NEC cases in Western countries has increased steadily from 1973 to 2012. The incidence in the United States in 2012 reached 6.98 per 100,000 people, compared to only 1.09 per 100,000 people in 1973, indicating that the incidence increased six-fold. Among 45,318 patients with NENs and a clear histological grade, the proportion of patients with NECs is as high as 32.6% (14,766/45,318) [4] . Studies have shown that 50% of patients with NECs have distant metastases at the time of diagnosis, and the median overall survival (OS) is only 5.8 months [4] . Treatments for advanced pulmonary NECs are mainly based on systemic therapies including chemotherapy, targeted therapy, and immunotherapy.
Advanced pulmonary NECs have a high degree of malignancy, relatively few diagnosis and treatment measures, and a poor prognosis. SCLC, which is the most common form, is still mainly treated with chemotherapy. Hence, this study analyzed the clinicopathological data and survival data of 123 patients with advanced intra-and extra-pulmonary NECs and summarized the clinical experience with the goal of providing guidance for the clinical treatment of SCLC.

Patients
One hundred-twenty-three patients with advanced intra-and extra-pulmonary NECs seen at the Fujian Medical University Union Hospital, Fujian Province, China, from January 2013 to November 2019 were included in this study. Data from the relevant cases were obtained from the case retrieval system of the medical record room of the Fujian Medical University Union Hospital. The inclusion criteria included: (1) diagnosed with NECs through histopathological examination of biopsy and dissected tumors. Patients had not received any prior chemotherapy, radiotherapy, and other treatments; and (2) with complete clinicopathological data. The exclusion criteria included: (1) combined with other types of malignant tumors; and (2) with medullary thyroid carcinoma, paraganglioma, or Merkel cell carcinoma.

Data collection
Clinical data meeting the inclusion criteria, such as the primary site of the tumor, age, gender, clinical symptoms, and treatment status were collected from pathological and clinical electronic medical records and subjected to statistical analysis.

Follow up
The survival and disease conditions of the 123 patients were followed up by case review and telephone follow-up. The deadline for follow-up was November 30, 2020. The survival of patients was presented in months and de ned as the duration from the date of diagnosis to the date of death or the end of followup. The observation endpoint of this study was the death of the patient and the observation index was OS. All surviving patients and patients lost to follow-up as of the last follow-up date were treated as censored data.

Statistical analyses
This study used the SPSS22.0 software package (IBM, Armonk, NY) for statistical analysis. The count data are presented as number of cases, and percentages and were compared by Chi-square test. Kaplan-Meier method and log-rank test were used for univariate survival analysis to calculate and compare survival rates. Signi cant variables in the univariate analysis were subjected to multivariate analysis using the Cox risk regression model. The level of effectiveness was α = 0.05.

Age
The median age of onset of patients with advanced pulmonary NECs was 59 years (ranging from 27 to 88 years), and the proportion of patients < 60 years old and ≥ 60 years old was 53.7% (66/123) and 46.3% (57/123), respectively. The median age of onset of intra-pulmonary NECs was 59 years (ranging from 27 to 88 years), and the proportion of patients < 60 years old and ≥ 60 years old was 52.2% (47/90) and 47.8% (43/90), respectively. The median age of onset of SCLC was 60 years (ranging from 27 to 88 years), and the proportion of patients < 60 years old and ≥ 60 years old was 48.1% (39/81) and 51.9% (42/81), respectively. The median age of onset for intra-pulmonary NECs other than SCLC (other intrapulmonary NECs) was 55 years (ranging from 41 to 59 years), and the proportion of patients < 60 years old and ≥ 60 years old was 100.0% (5/5) and 0.0% (0/5), respectively. The median age of onset in the extra-pulmonary NECs group was 58 years (ranging from 31 to 78 years), and the proportion of patients < 60 years old and ≥ 60 years old was 57.6% (19/33) and 42.4% (14/33), respectively. The median age of onset of extra-pulmonary NECs in the gastrointestinal tract was 59 years (ranging from 32 to 74 years), and the proportion of patients < 60 years old and ≥ 60 years old was 56.0% (14/25) and 44.0% (11/25), respectively. The median age of onset of extra-pulmonary NECs in other regions was 57 years (ranging from 31 to 78 years), and the proportion of patients < 60 years old and ≥ 60 years old was 62.5% (5/8) and 37.5% (3/8), respectively (Table 1, Figure 1). Compared with the SCLC patients who received chemotherapy alone, the median OS of SCLC patients who received combined chemotherapy and radiotherapy was signi cantly prolonged (P < 0.05). The median OS of patients that received ≥ 4 cycles of rst-line chemotherapy was signi cantly higher than patients that received < 4 cycles of rst-line chemotherapy (P < 0.05, Table 2). Results of univariate analysis affecting prognosis Univariate analysis of age, primary tumor site, albumin, hemoglobin, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), liver metastasis, bone metastasis, brain metastasis, tumor proliferation index, tissue morphology, number of cycles of rst-line chemotherapy, and chest radiotherapy showed that age, primary tumor site, ALP, LDH, liver metastasis, brain metastasis, number of cycles of rst-line chemotherapy, and chest radiotherapy were signi cantly correlated with the OS of patients with NECs (P < 0.05). Alternatively, sex, albumin, hemoglobin, bone metastasis, tumor proliferation index, and histological morphology were not signi cantly correlated with the OS of patients with NECs (P > 0.05, Table 2).

Results of multivariate analysis affecting prognosis
Cox regression analysis of eight variables including age, tumor primary site, ALP, LDH, liver metastasis, brain metastasis, number of cycles of rst-line chemotherapy, and chest radiotherapy showed that age, liver metastasis, number of cycles of rst-line chemotherapy, and chest radiotherapy were signi cantly correlated with the OS of patients with NECs (P < 0.05, Table 3, Figure 2). poorly differentiated NECs [9] . NECs are de ned as high-grade tumors with high malignancy, poor prognosis, and lack of speci c clinical features.
To date, the common locations of NECs in the Asian population are the lung, unknown primary site, cervix, pancreas, rectum, stomach, and bladder, according to the evidence-based medicine data of the Surveillance, Epidemiology, and End Results (SEER) database in the United States [5] . According to the statistical data of this study, the lung was the site with the highest incidence of NECs, followed by the esophagus, stomach, pancreas, mediastinum, liver, and rectum, which is different from reports from other countries. We speculate that this may be due to the small sample size in this study, which failed to fully summarize the features of the primary site of NECs in China. The median age of NECs in this study was 59 years old, which is consistent with the conclusions of other studies in China [10,11] , while a foreign study showed that the median age of patients with NECs was 67 years old [5] . The differences in patient median age may be due to racial differences. Previous studies have shown that the incidence of NECs in both men and women is equal [5,12] , while in this study, the incidence of NECs in males was higher than that of females. In addition, the incidence of intra-pulmonary NECs in males was also higher than in females, which is consistent with the ndings reported by Ichiki et al. [13] . Leone et al. showed that lung cancer is associated with smoking [14] , and a study by Bernhardt et al. showed that SCLC was signi cantly correlated with smoking [15] . A study on smoking in China showed that the smoking rate in Chinese males was signi cantly higher than that of Chinese females [16] . Therefore, smoking may be an important factor leading to the high incidence of intra-pulmonary NECs in males in this study.
Currently, there is no uniform treatment standard or norms for NECs, and the treatment principles are mainly based on the primary site of the tumor [19,20] . The main pathological types of NECs in the advanced lung are SCLC and LCNEC. The treatment for advanced SCLC remains chemotherapy. According to the NCCN Guidelines for cancer diagnosis and treatment, the rst-line chemotherapy for advanced SCLC is 4-6 cycles of etoposide combined with cisplatin (EP). There is debate on the therapeutic outcomes between irinotecan with combined cisplatin (IP) and EP regimens. In 2002, Japanese researchers used the IP regimen for treatment of patients with extensive SCLC and showed an improved total effective rate (65% vs. 52%, P = 0.02) and survival rate (12.8 months vs. 9.4 months, P = 0.002) [21] . According to the S0124 randomized phase III clinical trial published by the American Society of Clinical Oncology, no signi cant differences in OS, progression-free survival (PFS), or effective rate were found between the EP and IP regimens. The S0124 trial did not con rm the results reported by the above Japanese group. In the clinical treatment of advanced SCLC, programmed cell death-1 (PD-1) and T lymphocyte programmed death-ligand 1 (PD-L1) checkpoint inhibitors showed high clinical activity. The IMpower133 study of the e cacy and safety of Atezolizumab combined with chemotherapy for extensive-stage SCLC showed that patients receiving Atezolizumab combined with chemotherapy had a signi cantly longer median OS (P = 0.0154) than patients receiving standard treatment. In addition, the median PFS of the patients that received Atezolizumab combined with chemotherapy was extended from 4.3 months to 5.2 months, suggesting that the combination of Atezolizumab and chemotherapy as a rst-line treatment achieved signi cant improvement in the survival of patients with extensive-stage SCLC [22,23] . The CASPIAN study used the PD-L1 inhibitor Durvalumab combined with chemotherapy as the rst-line treatment of extensive-stage SCLC and showed that this combination therapy signi cantly increased the median OS (P = 0.0047) and lowered the mortality of the patients compared with the patients receiving standard treatments [24] .
The univariate analysis in this study showed that tissue morphology was not correlated with the OS of patients with NECs. Previous studies have shown that patients with pulmonary LCNEC have better prognosis than those with SCLC. In our study, the median OS of patients with other intra-pulmonary NECs was 27.07 months, which was longer than that of patients with SCLC (12.97 months), although no signi cant difference was found between the two groups of patients. This is possibly related to the small sample size of the other intrapulmonary NECs. According to statistics from the US SEER database, the median OS of advanced intra-pulmonary NECs is 5.8 months, the median OS of extra-pulmonary NECs in the gastrointestinal tract is 5.2 months, the median OS of extra-pulmonary NECs in other regions is 7.5 months, and the median OS of extra-pulmonary NECs in other regions is longer than that of intrapulmonary NECs [5] . In addition, a study by Lokesh et al. showed that the median OS of extra-pulmonary NECs was longer than that of intra-pulmonary NECs (13 months vs. 8 months) [40] . In our study, however, the median OS of intra-pulmonary NECs (13.53 months) was signi cantly longer than that of extrapulmonary NECs in the gastrointestinal tract (9.42 months) and extra-pulmonary NECs in the other regions (8.69 months). The difference between our study and the other studies may be due to the different populations. In addition, compared with extra-pulmonary NECs, intra-pulmonary NECs have been studied more extensively, and their diagnosis and treatment are more systematic and standardized.
Multivariate analysis in this study showed that age, liver metastasis, the number of cycles of rst-line chemotherapy, and chest radiotherapy were risk factors affecting median OS in patients with NECs. In terms of age, the data from this study suggests that young patients are more likely to bene t from treatment than older patients. Previous studies by Yao et al. and Yucel et al. also showed a correlation between survival and patient age in patients with NECs [12,41] . The liver is the most common metastasis site in patients with NECs. After liver metastasis occurs, tumor progression increases, which has an enormous impact on the quality of life and prognosis of the patient. In this study, results of multivariate analysis indicated that liver metastasis as an independent prognostic factor affecting the median OS of NECs patients, which is consistent with the ndings of many previous studies [42][43][44][45] . Studies have shown no statistical difference in improvement in prognosis or survival rate between a regimen of 4-6 cycles of chemotherapy and > 6-cycle chemotherapy. A larger number of chemotherapy cycles increases toxicity. Therefore, 4-6 cycles are generally used during platinum-based chemotherapy for NECs. In this study, patients receiving ≥ 4 cycles of chemotherapy had a signi cantly improved median OS compared with those receiving < 4 cycles of chemotherapy, which is consistent with the ndings of a previous study [46] . The more cycles of rst-line chemotherapy, the longer the disease progression-free time, which is equivalent to prolonging OS. Although the survival of patients with SCLC is signi cantly improved by chemotherapy, 80% of these patients are prone to intrathoracic recurrence. One study showed that after three cycles of EP treatment in advanced SCLC patients, patients with complete remission of chest lesions and patients with complete or partial remission of intrathoracic lesions had a signi cant increase in survival after receiving thoracic radiotherapy [47] . In this study, 15 patients with SCLC received combined radiotherapy and chemotherapy, and the survival bene t was signi cantly increased compared with patients receiving chemotherapy alone (19.83 months vs. 9.93 months), which is consistent with previous reports.
In conclusion, the prognosis of advanced intra-and extra-pulmonary NECs is poor, especially for SCLC, which is the dominant intra-pulmonary NEC and commonly requires chemotherapy. Comprehensively integrating age, presence or absence of liver metastasis, and other aspects of patient information is necessary to diagnosis and treatment. In addition, following the principle of individualization and providing the best or most appropriate diagnosis and treatment is required under the standardized framework. Given the unique heterogeneity of SCLC, scholars have found that there are still many questions to be answered in the eld of drug therapy, and more basic research and clinical research are needed to provide answers.

Declarations
Acknowledgements None Authors' contributions Chen Xiaoyun designed the study and drafted the manuscript. Guo Peilin contributed to the data collection and the analysis of the data. Yang Fan helped in data analysis. Chen Xiangqi and Yang Sheng revised the manuscript and approved the nal version for publication. All authors have read and approved the nal manuscript.

Funding
This work was supported by the Fujian Provincial Health Technology Project(2020CXB016).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable. Effects of age, liver metastasis, number of cycles of rst-line chemotherapy, and chest radiotherapy on OS in patients with NECs