NPM1 is a prognostic biomarker and correlated with clinicopathological characteristics in gastric cancer

doi:10.21203/rs.3.rs-1323441/v1

Background and aims: NPM1 plays an important role in the occurrence and development of leukemia, and its role in some solid tumors has gradually received attention. The purpose of this study is to explore the expression of NPM1 in gastric cancer (GC) tissues, to study the relationship between NPM1 expression and clinicopathological characteristics of GC patients, and to study the influence of NPM1 expression levels on the prognosis of GC patients.

Methods: The study was conducted from tissue samples obtained after radical gastrectomy in 106 patients with GC. We used tissue microarray immunohistochemical analysis to examine the expression level of NPM1 in different GC and adjacent tissues, and finally analyzed the relationship between NPM1 expression, clinicopathological factors and survival rate.

Results: Compared with the corresponding adjacent tissues, NPM1 is low-expressed in 75.5% of GC tissues. High NPM1 expression group was associated with better overall survival rate and disease free survival rate compared to low NPM1 expression group with GC (p<0.01).

Conclusion：The assessment of NPM1 expression in GC samples may represent a useful tool for GC diagnosis and prognostic prediction. Nonetheless, our study should be regarded as a preliminary study, and its predictive value as a single-center retrospective study is limited.

Gastric cancer

Nucleophosmin1

Tissue microarray

Overall survival

Disease free survival

Gastric cancer(GC) is the most common gastrointestinal tumor in the world and one of the main causes of cancer-related deaths[1, 2]. Early GC is asymptomatic or non-specific, and the diagnosis is often at an advanced stage, resulting in a low 5-year survival rate[3-6]. The incidence of GC has obvious regional characteristics, it is more likely to occur in East Asia and the mortality rate is high[7]. Personalized treatment for GC based on markers is considered as one of the methods to improve the 5-year overall survival (OS) rate of patients with GC. Finding new molecular biomarkers for GC is of great significance to improve the level of diagnosis and treatment[8, 9].

Nucleophosmin1 (NPM1, also known as B23) is a nucleolar shuttle phosphoprotein containing 294 amino acids[10]. It can quickly shuttle between the cytoplasm and the nucleus. This function determines that NPM1 can participate in many cells biological processes and perform different functions[11]. For example, it can be used as a molecular chaperone to regulate the function of histones[12]. It can also regulate the assembly of ribosomes[13, 14], DNA repair[15, 16] and cell apoptosis[17, 18] and so on. Over the years, numerous studies have shown that NPM1 can cause acute myeloid leukemia in the case of mutation or continuous high expression, and it plays a vital role in the occurrence and development of acute myeloid leukemia[19-21]. NPM1 can also participate in the occurrence and development of a variety of solid tumors at the same time[10, 22, 23].

So far, there are few studies on the relationship between NPM1 and GC, and controversy still exists between the research results. One study reported that expression level of NPM1 was significantly reduced in GC samples compared to matched non-tumor tissues samples and the low expression of NPM1 is obviously related to the distant metastasis of GC [24]. Another study reported that NPM1 expression significantly higher in GC tissues than in adjacent noncancerous tissues and the expression rates of NPM1 was significantly higher in patients with distant metastasis and more advanced tumor stages[25]. In view of the important role of NPM1 in neoplastic diseases and the recent contradictory research status in gastric cancer, it is necessary to do further research on its role and mechanism in gastric cancer. In this study, tissue microarray was used to analyzed NPM1 protein expression between GC tissues and matched noncancerous gastric samples. We also assessed the possible association between NPM1 and different clinicopathological features. Furthermore, we analyzed and evaluated the impact of NPM1 expression level on the prognosis of gastric cancer patients. Finally, a receiver operating characteristic (ROC) curve was generated to investigate the biomarker potential of NPM1 in GC prognostic diagnosis.

Patients

This study was approved by the research ethics committee at the Lanzhou University Second Hospital (NO:2021A-561). One hundred and six patients with primary GC that underwent surgical resection from Department of general surgery of Lanzhou University Second Hospital between January 2015 and October 2016 were adopted in this study. The recruited criteria: 1) A histological diagnosis of gastric adenocarcinoma. 2)The patients age ≥18 yeas. 3) Patients undergoing surgical resection of primary GC. 4) All data about pathological, treatment, surgical, and follow-up is complete. 5) The patient has signed an informed consent. The exclusion criteria: 1) Cases that died before discharge. 2) Receipt of preoperative chemoradiation or neoadjuvant chemotherapy, and 3) Multiple cancers patients within five years. Of the 106 patients in cohort, 79 had received standard adjuvant postoperative chemotherapy (5-fluorouracil or oxaliplatin-based regimen) within first month after surgery, and 27 patients had not due to the financial reasons. None of them accepted neoadjuvant chemotherapy or perioperative chemoradiation. Tumors were histologically staged according to the 7th edition of the TNM classification by the American Joint Committee on Cancer (AJCC). All patients participating in this research understand the content of this study and agree to participate in it.

Follow-up is conducted through normal outpatient clinics and telephone. The follow-up time is 2 weeks after discharge, once every 3 months in the first and second years, and 6 months in the following 3 years. The overall survival time is the time interval from surgery to the end of follow-up or death. Disease-free survival (DFS) time refers to the time interval between the first recurrence or the end of the follow-up during the postoperative follow-up period.

Tissue microarray construction

Tissue microarray was built from 106 patients with excised specimens of primary gastric tumors. The hematoxylin and eosin stained pathological sections of the included cases were read, and the H-E stained pathological sections and the representative positions on the corresponding wax blocks were marked. Use a puncher to punch holes on the sample wax block to obtain tissue columns, and load them in the chip wax block according to the sequence of arrangement. Each sample has 3 multiple points.

Immunohistochemical staining

Use immunohistochemical staining analysis according to the procedure previously described by the manufacturer[26]. For antigen retrieval, the TMA slides are dewaxed, rehydrated and boiled in a pressure pot with sodium citrate buffer (pH 6.0). The TMA slides were blocked with inhibitor (3% hydrogen peroxide) for thirty min at 37˚C after antigen retrieval. Use NPM1 antibody dilution for immunohistochemical staining (NPM1,1:200, Abcom, Cambridge, USA). Use the primary antibody for 25 minutes at room temperature and overnight at 4°C. Then rewarm for 15 minutes, wash with TBS 3 times, add 50μL of secondary antibody, and incubate for 25 minutes at room temperature. Rinse 3 times with TBS, DAB develops color, hematoxylin counterstain, rinse with water, dehydrate, and mount the film. Observe the expression of NPM1 protein under an optical microscope and take photos of the pictures.

Evaluation of immunostaining

Under the microscope, the complete tissue structure can be observed and the brown-yellow particles with obvious distribution in the background cells are judged as positive. The immunoreactive score (IRS) method[27] was used for semi-quantitative scoring according to the degree of staining: 0=no, 1=light yellow, 2= brown, and 3= dark brown. Then the percentage of positively stained tumor cells in each field were calculated, and take the average value for score: the percentage of positive cells no is 0, less than 10% is 1, and 10%-50% is 2, 51%-80% is 3, more than 80% is 4. The two scores are multiplied: 0 is considered negative (0+), 1 to 4 are considered weakly positive (1+), 5 to 8 are considered moderately positive (2+), and 9 to 12 are considered strong positive (3+). If there are multiple visual fields with different scores in the same specimen, the average value of the maximum value and the minimum value is taken as the immunohistochemical score. All arrays were reviewed by two unsuspecting pathologists. Review and discuss the inconsistent cases again until a consensus reached.

Statistical analysis

The relationship between the clinicopathological characteristics and expression level of NPM1 were assessed using the Pearson’s chi-squared test (χ² test) or Fisher's exact probability test. Kaplan-Meier method was used to build OS and DFS curves. The significance of OS and DFS between the NPM1 high and low expression groups was tested by log-rank test. Cox proportional hazards regression model was used for univariate and multivariate survival analysis. The ROC curve is designed to evaluate the diagnostic value of NPM1 in GC. Each statistical test was two-sided, the difference is statistically significant when p<0.05. All statistical analyses were performed using SPSS 23.0 statistical software package (SPSS, Chicago, IL, USA).

The expression of NPM1 in gastric cancer and patient’s clinicopathological characteristics

Representative expression levels (0+, 1+, 2+, 3+) of NPM1 is shown in Figure 1. The cohort consisted of 83 (78.3%) male and 23 (21.7%) female, with a median patient age of 64 (range 31–88) years. Sixty seven percent of tumors are located in the antrum of the stomach, 22.6% are located in the body of the stomach, and about 10.4% are located at the junction of the cardia and the esophagus. The intestinal type and diffuse type in Lauren classification accounted for 56.6% and 43.4 % respectively. Noticeably, 42 cases (39.6%) were in stages Ib-II, 64 cases (60.4%) were in stages III-IV (according to the 7th edition of the TNM classification by AJCC). Seventy-nine patients had received standard adjuvant postoperative chemotherapy (5-fluorouracil or oxaliplatin-based regimen) within first month after surgery, and 27 patients had not due to the financial reasons. The clinicopathologic data and demographic features of the 106 GC patients are summarized in Table 1.

Relationship between clinicopathological factors and NPM1 expression

Fifteen clinicopathological factors of high and low expression of NPM1 were compared separately (Table 2). There are significant differences in the venous invasion and AJCC stage (p<0.05).

Relationship between NPM1 expression level and prognosis of GC

Kaplan-Meier curves for OS and DFS rates in NPM1 high and NPM1 low expression groups were shown in Figure 2. The OS rates in patients with high and low NPM1 expression were 49.3% and 33.1%, respectively (Figure 2A). Compared with the low NPM1 expression group, the high NPM1 expression group showed significantly better OS (by log-rank test p<0.05). The DFS rates in patients with high and low NPM1 expression was 34.3% and 19.3%, respectively (Figure 2B). Compared with the low NPM1 expression group, the high NPM1 expression group showed significantly better DFS (by log-rank test p<0.01).

Univariate and multivariate COX regression analyses of OS and DFS

In the univariate and multivariate COX regression analyses of OS, we analyzed: age, gender, location of tumor, tumor size, pT stage (pathological assessment of primary tumor), pN stage(pathological assessment of regional lymph nodes), AJCC stage, lymphatic invasion, Lauren’s classification, venous invasion, carcino-embryonic antigen (CEA), carbohydrate antigen199 (CA199), carbohydrate antigen724 (CA724), α-fetoprotein (AFP), carbohydrate antigen125 (CA125), and NPM1 expression. In the univariate analyses, age, tumor size, pT stage, AJCC stage, Venous invasion and NPM1 expression were selected as significant factors for OS. Meanwhile, in the multivariate analyses, age, tumor size, pT stage, AJCC stage, Venous invasion and NPM1 expression were independent predictors of OS in curative gastrectomy patients (Table 3).

In the univariate and multivariate COX regression analyses of DFS, we analyzed: age, gender, location of tumor, tumor size, pT stage, pN stage, AJCC stage, lymphatic invasion, Lauren’s classification, venous invasion, CEA, CA199, CA724, AFP, CA125, and NPM1 expression. In the univariate analyses, age, pT stage, pN stage AJCC stage, Venous invasion and NPM1 expression were selected as significant factors for DFS. Meanwhile, in the multivariate analyses, age, pT stage, pN stage, AJCC stage, Venous invasion and NPM1 expression were independent predictors of DFS in curative gastrectomy patients (Table 4).

Diagnostic accuracy of NPM1 for predicting the prognosis of patients with GC

The ROC curve for diagnostic accuracy of NPM1 for the predicting the prognosis of patients with GC is presented in Figure 3. NMP1 scores yielded an area under the curve (AUC) of 0.712 (95% CI, 0.616-0.818), with a sensitivity of 77.3% and a specificity 78.2%, a cutoff value at 2.0 points.

For a long time, numerous studies have shown that NPM1 can cause acute myeloid leukemia in the case of mutation or continuous high expression, and it plays a vital role in the occurrence, development and prognosis of acute myeloid leukemia[19-21]. NPM1 can also participate in the occurrence and development of a variety of solid tumors at the same time[10, 22, 23]. In oral squamous cell carcinoma, higher NPM1 expression is significantly associated with larger tumor size, lymph node metastasis, and advanced clinical stage[28]. Multivariate analysis results show that higher NPM1 is associated with a worse prognosis[28]. Another study shows that the expression level of NPM1 in lung adenocarcinoma samples was higher than that in adjacent normal paracancerous tissues. NPM1 has high specificity and sensitivity for the diagnosis and prognosis assessment of lung adenocarcinoma[29]. Recently study reports that NPM1 expression is significantly increased in colorectal cancer and is associated with a poorer 5-year overall survival rate[30]. Above studies are examples of the high expression of NPM1 in different tumors. Its high expression promotes the biological behavior of the tumor and predicts a poor prognosis. However, it is interesting that there are different reports showing that NPM1 is lower-expressed in some tumors. Its low expression promotes the biological behavior of tumors and predicts a poor prognosis. Luo etc.[31] reported that NPM1 has a low expression in bladder cancer cells, which is also related to the poor prognosis of bladder cancer. Knockdown of NPM1 expression in bladder cancer cell lines can significantly improve tumor cell migration and invasion capabilities. The silencing of NPM1 will accelerate the tumorigenicity of drug-resistant bladder cancer cells. Karhemo etc.[32] reported that NPM1 is low expressed in breast cancer, and the decrease of NPM1 protein level in breast cancer is related to poor prognosis. Histologically, the luminal epithelial cells of normal breasts showed high levels of NPM1 expression. The overexpression of NPM1 in breast cancer cells MDA-MB-231 stopped their growth in soft agar. NPM1 has a tumor suppressor effect in breast cancer. It can be seen that due to the different types of tumors, NPM1 plays different roles. It acts as a tumor-promoting factor in some tumors, but it acts as a tumor suppressor in other tumors.

So far, there are few studies on the relationship between NPM1 and GC, and controversy still exists between the research results. One study reported that expression level of NPM1 was significantly reduced in GC samples compared to matched non-tumor tissues samples and the low expression of NPM1 is obviously related to the distant metastasis of GC[24]. Another study reported that NPM1 expression significantly higher in GC tissues than in adjacent noncancerous tissues and the expression rate of NPM1 was significantly higher in patients with distant metastasis and more advanced tumor stages[25]. In view of the important role of NPM1 in neoplastic diseases and the recent contradictory research status in gastric cancer, it is necessary to do further research on its role in GC.

In this study, we used the tissue microarray method to detect the expression level of NPM1 in the GC tissues and adjacent normal tissues in 106 GC patients. The result of this study shows that in 75.5% of matched samples of gastric cancer and adjacent tissues, the NPM1 in gastric cancer samples was lower expressed than that of matched normal adjacent tissues. It can be seen that the expression level of NPM1 in most gastric cancer tissues is significantly lower than that of normal adjacent tissues.

Survival analysis results show that low expression of NPM1 was significantly associated with worse OS and DFS. The OS rate in patients with high and low NPM1 expression was 49.3% and 33.1%, respectively. Compared with the low NPM1 expression group, the high NPM1 expression group showed significantly better OS (by log-rank test p<0.05). The DFS rate in patients with high and low NPM1 expression was 34.3% and 19.3%, respectively. Compared with the low NPM1 expression group, the high NPM1 expression group showed significantly better DFS (by log-rank test p<0.01). It can be seen that the expression level of NPM1 has a direct impact on the OS and DFS in GC patients. NPM1 exists as a tumor suppressor in GC.

In the univariate COX regression analyses, age, tumor size, pT stage, AJCC stage, Venous invasion and NPM1 expression were selected as significant factors for OS. Meanwhile, in the multivariate COX regression analyses, age, tumor size, pT stage, AJCC stage, Venous invasion and NPM1 expression were independent predictors of OS in curative gastrectomy patients.

In the univariate COX regression analyses, age, pT stage, pN stage, AJCC stage, Venous invasion and NPM1 expression were selected as significant factors for DFS. Meanwhile, in the multivariate COX regression analyses, age, pT stage, pN stage, AJCC stage, Venous invasion and NPM1 expression were independent predictors of DFS in curative gastrectomy patients.

The ROC curve for diagnostic accuracy of NPM1 for the predicting the prognosis of patients with GC was demonstrated. NMP1 scores yielded an AUC of 0.712 (95% CI, 0.616-0.818), with a sensitivity of 77.3% and a specificity 78.2%, a cutoff value at 2.0 points. The assessment of NPM1 expression in GC samples may represent a useful tool for GC diagnosis and prognostic prediction.

Our research has some limitations. In the first place, although the number of GC patients in this study is relatively large (n=106), this is a single-center, retrospective study. In order to obtain a more reliable analysis of the clinical significance of NPM1 in GC, a multicenter study includes a larger number of patients is also needed. In the second place, we used immunostaining to examine the expression of NPM1 in the central and peripheral parts of each GC tissue; however, considering the heterogeneity, the expression level of NPM1 at the sampling site may not be representative of the entire tumor area.

The assessment of NPM1 expression in GC samples may represent a useful tool for GC diagnosis and prognostic prediction. Nonetheless, our study should be regarded as a preliminary study, and its predictive value as a single-center retrospective study is limited.

Ethics approval and consent to participate

This study was approved by the research ethical review board at the Lanzhou University Second Hospital (NO:2021A-561). All experiments were performed in accordance with relevant guidelines and regulations. All participants in this study (or their parents/legal guardians/close relatives) obtained written informed consent to participate in the study.

Consent for publication

All authors have given their consent for publication in this journal.

Availability of data and materials

All data generated or analyzed during this study are included in this article. For further inquiries, please contact the corresponding author directly. There are no multimedia and supplementary materials. Each file not exceed 10 MB in size.

Competing interests

Authors declare that they have no conflict of interest.

Funding

This research was supported by the Gansu Provincial Natural Science Foundation Project (21JR11RA106) and the Gansu Provincial Youth Science and Technology Fund Project (21JR7RA424).

Author Contributions

CAG and HBL conceived and designed the study. XLS, THX, XMC, and SBY extracted and collected the data. XLS, AZ, and WAW analyzed the data. CAG, WJW, and XLS performed experiments. CAG, and AZ prepared figures and tables. CAG, XLS, and HBL wrote and revised the draft manuscript. All authors reviewed and approved the final manuscript.

Acknowledgements

There are no organizations that have made substantive contributions to the research.

Author details

1. Second Clinical Medical College, Lanzhou University, Lanzhou, Gansu Province, China.

2. Department of Pathology, Lanzhou University Second Hospital, Lanzhou, Gansu Province, China.

3. Department of general surgery, Lanzhou University Second Hospital, Lanzhou, Gansu Province, China.

4. Clinical Medical College, Ningxia Medical University, Yinchuan, the Ningxia Hui Autonomous Region, China.

5. Clinical Medical College, Gansu University of Chinese Medicine, Lanzhou, Gansu Province, China.

Siegel RL, Miller KD, Fuchs HE, et al. Cancer statistics, 2021. CA Cancer J Clin 2021;71(1): 7-33.
Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021;71(3): 209-249.
Venerito M, Vasapolli R, Rokkas T , et al. Gastric cancer: Epidemiology, prevention, and therapy. Helicobacter 2018;23 Suppl 1(e12518.
Xie JW, Lu J, Xu BB, et al. Prognostic value of tumor regression grading in patients treated with neoadjuvant chemotherapy plus surgery for gastric cancer. Front Oncol 2021;11(587856.
Bae JS, Chang W, Kim SH, et al. Development of a predictive model for extragastric recurrence after curative resection for early gastric cancer. Gastric Cancer 2021.
Eloranta S, Smedby KE, Dickman PW, et al. Cancer survival statistics for patients and healthcare professionals - a tutorial of real-world data analysis. J Intern Med 2021;289(1): 12-28.
Hong S, Won YJ, Lee JJ, et al. Cancer statistics in korea: Incidence, mortality, survival, and prevalence in 2018. Cancer Res Treat 2021;53(2): 301-315.
Pandey I, Misra V, Pandey AT, et al. Artificial intelligence technologies empowering identification of novel diagnostic molecular markers in gastric cancer. Indian J Pathol Microbiol 2021;64(Supplement): S63-S68.
Molinari C, Tedaldi G, Rebuzzi F, et al. Early gastric cancer: Identification of molecular markers able to distinguish submucosa-penetrating lesions with different prognosis. Gastric Cancer 2021;24(2): 392-401.
Grisendi S, Mecucci C, Falini B, et al. Nucleophosmin and cancer. Nat Rev Cancer 2006;6(7): 493-505.
Box JK, Paquet N, Adams MN, et al. Nucleophosmin: From structure and function to disease development. BMC Mol Biol 2016;17(1): 19.
Okuwaki M, Matsumoto K, Tsujimoto M, et al. Function of nucleophosmin/b23, a nucleolar acidic protein, as a histone chaperone. FEBS Lett 2001;506(3): 272-276.
Maggi LB, Jr., Kuchenruether M, Dadey DY, et al. Nucleophosmin serves as a rate-limiting nuclear export chaperone for the mammalian ribosome. Mol Cell Biol 2008;28(23): 7050-7065.
Pozzo F, Bittolo T, Vendramini E, et al. Notch1-mutated chronic lymphocytic leukemia cells are characterized by a myc-related overexpression of nucleophosmin 1 and ribosome-associated components. Leukemia 2017;31(11): 2407-2415.
Lopez DJ, Rodriguez JA and Banuelos S. Nucleophosmin, a multifunctional nucleolar organizer with a role in DNA repair. Biochim Biophys Acta Proteins Proteom 2020;1868(12): 140532.
Lopez DJ, de Blas A, Hurtado M, et al. Nucleophosmin interaction with ape1: Insights into DNA repair regulation. DNA Repair (Amst) 2020;88(102809.
Li S, Zhang X, Zhou Z, et al. Downregulation of nucleophosmin expression inhibited proliferation and induced apoptosis in salivary gland adenoid cystic carcinoma. J Oral Pathol Med 2017;46(3): 175-181.
Jeon YJ, Cho JH, Lee SY, et al. Esculetin induces apoptosis through egfr/pi3k/akt signaling pathway and nucleophosmin relocalization. J Cell Biochem 2016;117(5): 1210-1221.
Falini B, Sciabolacci S, Falini L, et al. Diagnostic and therapeutic pitfalls in npm1-mutated aml: Notes from the field. Leukemia 2021.
Falini B, Brunetti L and Martelli MP. How i diagnose and treat npm1-mutated aml. Blood 2021;137(5): 589-599.
Zarka J, Short NJ, Kanagal-Shamanna R, et al. Nucleophosmin 1 mutations in acute myeloid leukemia. Genes (Basel) 2020;11(6).
Lim MJ and Wang XW. Nucleophosmin and human cancer. Cancer Detect Prev 2006;30(6): 481-490.
Karimi Dermani F, Gholamzadeh Khoei S, Afshar S, et al. The potential role of nucleophosmin (npm1) in the development of cancer. J Cell Physiol 2021.
Leal MF, Mazzotti TK, Calcagno DQ, et al. Deregulated expression of nucleophosmin 1 in gastric cancer and its clinicopathological implications. BMC Gastroenterol 2014;14(9.
Zhou F, Chen E, You D, et al. Both high expression of nucleophosmin/b23 and crm1 predicts poorer prognosis in human gastric cancer. APMIS 2016;124(12): 1046-1053.
Gu J, Zhang S, He X, et al. High expression of pig11 correlates with poor prognosis in gastric cancer. Exp Ther Med 2021;21(3): 249.
Segami K, Aoyama T, Hiroshima Y, et al. Clinical significance of tap1 and dll4 expression in patients with locally advanced gastric cancer. In Vivo 2021;35(5): 2771-2777.
Peng HH, Ko HH, Chi NC, et al. Upregulated npm1 is an independent biomarker to predict progression and prognosis of oral squamous cell carcinomas in taiwan. Head Neck 2020;42(1): 5-13.
Zhou LM, Yuan LL, Gao Y, et al. Nucleophosmin 1 overexpression correlates with (18)f-fdg pet/ct metabolic parameters and improves diagnostic accuracy in patients with lung adenocarcinoma. Eur J Nucl Med Mol Imaging 2021;48(3): 904-912.
Yu ACY, Chern YJ, Zhang P, et al. Inhibition of nucleophosmin 1 suppresses colorectal cancer tumor growth of patient -derived xenografts via activation of p53 and inhibition of akt. Cancer Biol Ther 2021;22(2): 112-123.
Luo C, Lei T, Zhao M, et al. The role of npm1 in the invasion and migration of drug resistant bladder cancer. Urol J 2021;18(4): 452-459.
Karhemo PR, Rivinoja A, Lundin J, et al. An extensive tumor array analysis supports tumor suppressive role for nucleophosmin in breast cancer. Am J Pathol 2011;179(2): 1004-1014.

Table 1

Clinicopathological characteristics of GC patients.

Variables

n=106

Age, median (range)

64(31-88)

Gender

Male

Female

83(78.3%)

23(21.7%)

Tumor location

Antrum

Body

Cardia

71(67.0%)

24(22.6%)

11(10.4%)

Tumor size

≥5cm

<5cm

48(45.3%)

58(54.7%)

pT stage

T1-T3

T4

40(37.7%)

66(62.3%)

Lymph-node metastasis

Positive

Negative

86(81.1%)

20(18.8%)

Lymphatic invasion

Positive

Negative

78(73.6%)

28(26.4%)

pN stage

N0

N1

N2

N3

20(18.8%)

19(17.9%)

26(24.5%)

41(38.7%)

Lauren’s classification

Intestinal type

Diffuse type

60(56.6%)

46(43.4%)

AJCC stage

Ib-II

III-IV

42(39.6%)

64(60.4%)

Venous invasion

Positive

Negative

73(68.9%)

33(31.1%)

CEA

Positive

Negative

58(55.0%)

48(45.0%)

CA199

Positive

Negative

44(41.5%)

62(58.5%)

CA724

Positive

Negative

52(49.1%)

54(50.9%)

AFP

Positive

Negative

11(10.4%)

95(89.6%)

CA125

Positive

Negative

22(20.8%)

84(79.2%)

NPM1 expression

Low

High

80(75.5%)

26(24.5%)

Adjuvant chemotherapy

Yes

No

79(74.5%)

27(25.5%)

Abbreviations: NPM1, nucleophosmin1. pT stage, pathological assessment of primary tumor. pN stage, pathological assessment of regional lymph nodes. CEA, carcino-embryonic antigen. CA199, carbohydrate antigen199. CA724, carbohydrate antigen 724. AFP, α-fetoprotein. CA125, carbohydrate antigen 125.

Table 2

Comparison of clinicopathological parameters between NPM1 negative and positive gastric cancer patients.

NPM1 expression

Low(n=80)

High(n=26)

p

Age (years), median (range)

66(32-88)

65(31-87)

0.102

Gender

Male

Female

63(78.8%)

17(21.2%)

20(76.9%)

6(23.1%)

0.158

Tumor location

Antrum

Body

Cardia

56(70.0%)

16(20.0%)

8(10.0%)

15(57.7%)

8(30.8%)

3(11.5%)

0.154

Tumor size

≥5cm

<5cm

36(45.0%)

44(55.0%)

12(46.2%)

14(53.8%)

0.149

Lauren’s classification

Intestinal type

Diffuse type

46(57.5%)

34(42.5%)

14(53.8%)

12(46.2%)

0.264

pT stage

T1-T3

T4

29(36.3%)

51 (63.7%)

11(42.3%)

15(57.7%)

0.056

pN stage

N0

N1

N2

N3

15(18.8%)

14(17.5%)

20(25.0%)

31(38.8%)

5(19.2%)

6(23.1%)

10(38.5%)

0.325

AJCC stage

Ib-II

III-IV

26(32.5%)

54(67.5%)

16(61.5%)

10(38.5%)

0.010

Venous invasion

Positive

Negative

64(80.0%)

16(20.0%)

9(34.6%)

17(65.4%)

0.023

Lymphatic invasion

Positive

Negative

60(75.0%)

20(25.0%)

18(69.2%)

8(30.8%)

0.259

CEA

Positive

Negative

42(52.5%)

38(47.5%)

16(61.5%)

10(38.5%)

0.092

CA199

Positive

Negative

33(41.3%)

47(58.7%)

11(42.3%)

15(57.7%)

0.328

CA724

Positive

Negative

40(50.0%)

12(46.2%)

14(53.8%)

0.219

AFP

Positive

Negative

8(10.0%)

72(90.0%)

3(11.5%)

23(88.5%)

0.146

CA125

Positive

Negative

16(20.0%)

64(80.0%)

6(23.1%)

20(76.9%)

0.287

Abbreviations: NPM1, nucleophosmin1. pT stage, pathological assessment of primary tumor. pN stage, pathological assessment of regional lymph nodes. CEA, carcino-embryonic antigen. CA199, carbohydrate antigen199. CA724, carbohydrate antigen 724. AFP, α-fetoprotein. CA125, carbohydrate antigen 125.

Table 3

Univariate and multivariate COX regression analyses of OS predictors in patients with GC (n=106).

Variable		Univariate COX regression						Multivariate COX regression
Variable	N	HR		95% CI		p-value		HR	95% CI	p-value
Age (years) <70 ≥70	33 73		2.369		1.733-3.046		0.015	2.254	1.856-2.965	0.025
Gender Male Female	83 23		0.761		0.463-1.214		0.254
Location of tumor Antrum Non-antrum	71 35		1.062		0.743-1.325		0.124
Tumor size ≥5cm <5cm	48 58		2.354		1.698-3.021		0.002	1.938	1.249-2.367	0.003
pT stage T1-T3 T4	40 66		1.785		1.245-2.021		0.023	1.865	1.256-2.214	0.005
pN stage N0 N1-N3	20 86		1.564		1.021-1.986		0.156
AJCC stage Ib-II III-IV	42 64		2.325		1.654-3.021		0.029	2.158	1.433-2.986	0.032
Lymphatic invasion Positive Negative	78 28		1.445		1.015-1.965		0.452
Lauren’s classification Intestinal type Diffuse type	60 46		1.251		0.856-1.834		0.091
Venous invasion Positive Negative	73 33		1.785		1.124-2.215		0.048	1.935	1.003-2.465	0.012
CEA Positive Negative	58 48		1.255		0.645-1.855		0.069
CA199 Positive Negative	44 62		0.966		0.542-1.455		0.122
CA724 Positive Negative	52 54		1.548		0.965-2.122		0.135
AFP Positive Negative	11 95		1.223		0.554-1.869		0.253
CA125 Positive Negative	22 84		0.965		0.258-1.562		0.114
NPM1 expression Low High	80 26		1.965		1.354-2.432		0.015	1.875	1.324-2.433	0.032
Abbreviations: NPM1, nucleophosmin1. pT stage, pathological assessment of primary tumor. pN stage, pathological assessment of regional lymph nodes. CEA, carcino-embryonic antigen. CA199, carbohydrate antigen199. CA724, carbohydrate antigen 724. AFP, α-fetoprotein. CA125, carbohydrate antigen 125. N, Number of patients. HR, Hazard ratio. CI, Confidence interval.

Table 4

Univariate and multivariate COX regression analyses of DFS predictors in patients with GC (n=106).

Variable

Univariate COX regression

Multivariate COX regression

N

HR

95% CI

p-value

HR

95% CI

p-value

Age (years)

<70

≥70

33

73

2.448

2.021-3.147

0.023

2.543

2.035-3.011

0.036

Gender

Male

Female

83

23

0.865

0.395-1.441

0.325

Location of tumor Antrum

Non-antrum

71

35

1.124

0.632-1.624

0.212

Tumor size

≥5cm

<5cm

48

58

1.533

0.744-2.125

0.062

1.993

1.147-2.452

0.089

pT stage

T1-T3

T4

40

66

1.663

1.114-2.142

0.012

1.965

1.145-2.359

0.011

pN stage

N0

N1-N3

20

86

1.978

1.154-2.534

0.014

1.895

1.126-2.433

0.026

AJCC stage

Ib-II

III-IV

42

64

2.547

1.553-3.154

0.035

2.248

1.536-3.024

0.022

Lymphatic invasion

Positive

Negative

78

28

1.665

1.125-2.012

0.325

Lauren’s classification

Intestinal type

Diffuse type

60

46

1.123

0.733-1.654

0.069

Venous invasion

Positive

Negative

73

33

1.865

1.235-2.354

0.032

1.754

1.102-2.214

0.021

CEA

Positive

Negative

58

48

1.356

0.725-1.965

0.126

CA199

Positive

Negative

44

62

1.133

0.423-1.654

0.232

CA724

Positive

Negative

52

54

1.446

1.325-1.968

0.253

AFP

Positive

Negative

11

95

1.354

0.785-1.954

0.154

CA125

Positive

Negative

22

84

1.032

0.368-1.625

0.091

NPM1 expression

Low

High

80

26

2.368

1.663-2.996

0.023

1.889

1.228-2.521

0.011

Abbreviations: NPM1, nucleophosmin1. pT stage, pathological assessment of primary tumor. pN stage, pathological assessment of regional lymph nodes. CEA, carcino-embryonic antigen. CA199, carbohydrate antigen199. CA724, carbohydrate antigen 724. AFP, α-fetoprotein. CA125, carbohydrate antigen 125. N, Number of patients. HR, Hazard ratio. CI, Confidence interval.

No competing interests reported.

NPM1 is a prognostic biomarker and correlated with clinicopathological characteristics in gastric cancer

Status:

Version 1

Abstract

Figures

Introduction

Methods

Patients

Tissue microarray construction

Immunohistochemical staining

Evaluation of immunostaining

Statistical analysis

Results

The expression of NPM1 in gastric cancer and patient’s clinicopathological characteristics

Relationship between clinicopathological factors and NPM1 expression

Relationship between NPM1 expression level and prognosis of GC

Univariate and multivariate COX regression analyses of OS and DFS

Diagnostic accuracy of NPM1 for predicting the prognosis of patients with GC

Discussion

Conclusion

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Author Contributions

Acknowledgements

Author details

References

Tables

Additional Declarations

Status:

Version 1