Integrated Analysis of Serum Lymphocytes Develops a Nomogram for Predicting Prognosis of Intracranial Germ Cell Tumors to Chemo-Radiotherapy

doi:10.21203/rs.3.rs-1447293/v1

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Integrated Analysis of Serum Lymphocytes Develops a Nomogram for Predicting Prognosis of Intracranial Germ Cell Tumors to Chemo-Radiotherapy

https://doi.org/10.21203/rs.3.rs-1447293/v1

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Introduction:

It is common to evaluate lymphocytes subsets in virus infection, immunodeficiency diseases and cancer. This study retrospectively analyzed serum lymphocyte subsets and survival outcomes in intracranial germ cell tumors (iGCTs) patients.

Methods

Serum from145 iGCTs patients were collected and lymphocyte subsets were analyzed by flow cytometry. Clinical information was extracted from the database in Sun Yat-sen University Cancer Center and outcomes were confirmed through outpatient visits and telephone follow-up.

Results

76 patients were confirmed germinoma, 29 mixed germ cell tumor, 4 teratoma, 9 malignant teratoma, 6 endodermal sinus tumor, 20 choriocarcinoma and 1 embryonal carcinoma. 123(84.8%) patients received comprehensive treatments and 104(71.7%)reached complete or partial response. T lymphocytes (CD3⁺), cytotoxic T cells (CD3⁺CD8⁺ or Tc) and B lymphocytes(CD19⁺)decreased(p = 0.033, p = 0.011, and p < 0.001 respectively), while activated cytotoxic T lymphocytes(CD8⁺CD25⁺) increased(p < 0.001). Dynamic changes of activated Tc cells were associated with treatment patterns and evaluation of the disease(p = 0.038 and p = 0.043). Median survival was 45.81 months and patients of increased Tc cells, activated T helper cells(CD4⁺CD25⁺T cells or activated Th cells), activated Tc cells and normal CD4⁺/CD8⁺ ratio presented encouraging outcomes (p = 0.007;0.016;0.028;0.016 respectively). Nomogram and multivariate Cox Regression analysis indicated increased Tc cells was related to reduced risk death(p = 0.001,HR = 0.217 95%CI = 0.085—0.551).)

Conclusion

Implement of comprehensive treatments led to positive responses. Dynamic monitoring of serum lymphocyte subsets can be used as an auxiliary indicator for prognosis judgment.

Intracranial germ cell tumors

Lymphocyte subsets

Prognosis

Adolescent tumors

Intracranial Germ Cell Tumors (iGCTs) are rare brain tumors that originate from embryonic germ cells. It is mainly seen in children and adolescents, with the highest incidence rate between 10–14 years old¹, the sellar region, pineal area and the basal ganglia area are the most commonly affected areas, especially the former two^2–4. The incidence rate varies greatly from continents: it is more common in Asia than in North America and Europe⁵: the total incidence rate in the United States is 0.6 per million per year, Europe is 1.0 per million per year, and Japan is 2.7 per million per year⁶. Classification of central nervous system germ cell tumors according to the World Health Organization divides them into germinoma and non-germinoma germ cell tumors(NGGCTs), with 6 different types of the latter, which includes teratoma, embryonal carcinoma, endodermal sinus tumor (yolk sac tumor), chorionic epithelioma (also called choriocarcinoma) and mixed germ cell tumors(GCTs)⁷(including above components sometimes teratoma and germinoma as well). Diagnostic methods vary in regions, some countries rely on surgical (a gross total resection or biopsy) and pathological verification whereas with consideration of safety and prognosis, others take tumor marker: α-fetoprotein (or AFP typically raised in the presence of yolk sac tumor) and human chorionic gonadotropin (HCG; typically raised in the presence of choriocarcinoma) together with steady radiological appearances and clinical characteristics as confirmation of diagnosis. Commonly, increased HCG is viewed to be positively correlated with poor prognosis⁸. However, it is ambiguous for simple tumor makers to evaluate patients’ changing conditions and to predict outcomes, not to mention the evaluation and prognostic significance of patients with negative tumor indicators. What’s more, though optimum management for iGCTs patients is recognized to form a collaborative team from multi-disciplines of experts, variable patterns increased the difficulty of personalized treatment decisions.

For recent decades, it has been recognized that malignant tumor cells potentially induce local and systemic immunosuppression⁹. Therefore the application of immunotherapy has driven the treatment of brain tumors to the study of the response of intratumoral and systemic immune cells and cytokines to these malignancies ^10–12. It is common to use lymphocytes subsets measurement in the evaluation of human immunodeficiency virus (HIV) infection¹³, primary immunodeficiency diseases¹⁴, autoimmune diseases¹⁵, adenocarcinoma¹⁶ and leukaemia ¹⁷. Studies confirmed that a large amount of activated CD4⁺CD25⁺ T cells played a crucial role in anti-tumor immune responses and were of beneficial prognostic influence in non-small cell lung carcinoma(NSCLC) patients¹⁸. On the contrary, increased CD4⁺CD25⁺ Treg cells related to augmenting of malignant cells in tumor microenvironment and high presence of CD25⁺ cells in peripheral blood was concerned of having reference to chemoresistance in lung cancer¹⁹, advanced renal carcinoma²⁰, breast cancer²¹, gastrointestinal malignancies²² and high virus load. Patients with higher intratunmoral or circulating Tc cells are prone to slower progression and live longer.²³ Accordingly, we speculate that an examination of the lymphocyte subsets dynamics has practical meaning for monitoring the progression and prognosis of intracranial germ cell tumors and helping with optimal clinical decision-making as an auxiliary indicator. Limited studies remain obscure in intracranial germ cell tumors immunomodulated status, further studies to understand the relationship of the tumor immune and the peripheral blood immune subsets with survival outcome are needed.

Patients: The ethics committee approved this retrospective study: 145 cases diagnosed as intracranial germ cell tumors and treated at Sun Yat-sen University Cancer Center from June 2011 till November 2019 were retrospectively collected. Patients with any of the following were excluded: patients with immunosuppression status, including autoimmune disease, post-operation of organ transplantation, and intaking immunosuppressive drug. Informed consent was obtained from all individual participants included in the study.
Treatment: Current comprehensive clinical management were customized for each patient as following listed until the development of unacceptable toxicity from therapies, withdrawal or death: a. platinum-based chemotherapy, etoposide, bleomycin and cisplatin, ifosfamide, and etoposide (EP, BEP or ICE)(carboplatin was recommended if patients suffered an adverse reaction to cisplatin), alternating at 21-days intervals with cycles; b. Surgery: tumor resections were primarily reserved for hypothesized iGCT patients with negative tumor markers, and ventricle-abdominal shunt or Gamma knife were suggested for those who could not tolerate tumor resection; c. Radiotherapy was administered after 4-6 cycles of chemotherapy, followed by additional two cycles of anti-tumor drugs. Regarding target volume, either whole-brain radiotherapy(WBNT) 21.6-36Gy or craniospinal irradiation(CSI) 19.8-30.6Gy with boost (16.2-23.4Gy) were considered. The gross target volume(GTV) was defined as the extent of the primary tumor(s) before treatment. The clinical target volume (CTV) was obtained by adding 1–2 cm to the GTV. For patients who suffered from progression, we provided extra temozolomide(TMZ) and Avastin.
Evaluation and Follow up: An evaluation was performed every 2 chemotherapy cycles with the following testing: a. Serum lymphocyte subsets and tumor maker were compulsive and those in CSF were performed for patients without contraindication. b. Lymphocyte subsets were counted by flow cytometry analysis. c. Criteria for Response Assessment Incorporating Magnetic Resonance Imaging and Clinical Factors (Supplementary Fig) was used for evaluation. Overall survival (OS) was defined from the time of first anti-tumor treatment to death from any cause or last follow-up.
Statistical analysis: IBM SPSS Statistics for Windows, version 25.0, and R, version 4.1.2 were used to perform the data analyses. Paired-sample t-test and one-way ANOVA, Wilcoxon test and Mann-Whitney U test, andX² test were used. The Kaplan Meier method was used to calculate the OS curves, and the log-rank test was employed to assess differences. Cox regression was applied to multivariate analysis. A two-sided p value< 0.05 was considered as the significance level.

Patient characteristics

A total of 145 patients were eligible for this study and their characteristics are summarized in Table 1. No statistical discrepancy was shown in patients’ demographics between tumor sites(p = 0.231), classification(p = 0.078) and outcomes (Table 1) Representative symptoms contains headache and vomiting(77/145), precocious puberty/abnormal menstruation in women/male libido(18/145), adipic diabetes insipidus(34/145), impaired vision(33/145), hemiparesis(29/145), epilepsy seizure(2/145) and other atypical symptoms(chest tightness, memory loss, hiccup singulation, fever and etc).

19 patients complained of recurrence of disease and seven of them had received surgery at the first visit. Time to progression(TTP) ranged from 4 to 98 months and the median TTP was 24 months. 2 patients who had undergone surgery did not perform follow-up chemotherapy and radiation treatment, which led to progression within 1 year. 3 patients completed the whole procedure of comprehensive therapy and lived over 24months TTP, 72 and 98 months for two of them, respectively. Neurohypophysis, pineal region, and basal ganglia regions are the most three prevalent areas⁵. Among the patients with recurrence of these diseases, the tumors of 6 patients were single lesions located in these common sites, and tumors of 7 violated the ventricle system. While only 2 patients whose tumors were bifocals(pineal gland and saddle) complained of recurrence.

The pattern of treatment details was summarized in Table 2. Regardless of comprehensive treatment models, chemotherapy or whether at least 6 cycles of chemotherapy were carried out, there was no significant relationship with the recurrence of the disease(p = 0.063,0.482,0.455, respectively). Treatment efficacy was completed through RANO criteria. Although different treatment patterns, chemotherapy regimens were not related to efficacy evaluation, whether radiotherapy combined with chemotherapy was related to different prognoses. Despite the correlation being fair.(p = 0.002, Camer’V = 0.365).

Comparison of Changes in Lymphocyte Subsets

The immune status could be distinguished pre and post chemotherapy. Serum lymphocytes including T(CD3⁺) cell, cytotoxic T cells(Tc or CD3⁺CD8⁺T cells), B(CD19⁺) cell significantly decreased(p = 0.033, p = 0.011 and p < 0.001),comparing with augment of activated cytotoxic T cells(activated Tc or CD8⁺CD25⁺ T cells)(p < 0.001) (Fig. 1A(a),(b),(c),(d)). Though, T helper cells(Th or CD3⁺CD4⁺ T cells), natural killer cells(NK cells or CD3^-CD16⁺CD56⁺ T cells) and activated T helper cells (activated Th cells or CD4⁺CD25⁺T cells) reduced in the consequence of chemotherapy, there was no significance(p = 0.713, p = 0.390 p = 0.276). (Fig. 1A(e),(f),(g)). These results indicated inducing one course of chemotherapy to patients with germ cell tumor could restrain the proliferation of immune cells, especially cell-mediated immunity.

Before treatments, the level of T cells and Tc cells in different tumor sites were discrepant(p = 0.0021 and 0.007respectively). Similarly, the distribution of T cells and B cells in different classifications of germ cells were statistically different(p = 0.014 and 0.028). Besides, the status of tumor markers in serum is also associated with types of germ cells. (p-value of AFP and β-hCG: p < 0.001 and p = 0.028).After one course of chemotherapy, whereas, immune status tended to be indifferent in various tumor sites and classifications, except humoral immunity, with a diverse distribution of B cells (p = 0.024).β-hCG in serum significantly and steadily implied statistical differences in different treatment methods, treatment courses whether combined with radiotherapy(p = 0.001, p < 0.001, p = 0.001).

To further explore which treatment mode caused differences in the level of lymphocyte subsets after treatment, we defined the patterns as comprehensive treatment (surgery + radiotherapy + chemotherapy), radio-chemotherapyand chemotherapy alone. We found that there was a statistically significant difference in B cells between the people who received chemotherapy alone and those with radio-chemotherapy(p = 0.021). Meanwhile, activated Tc cells were significantly different between the groups of comprehensive treatment and radio-chemotherapy(p = 0.015). The same results could be seen in the comprehensive treatment group and the chemotherapy alone group(p = 0.034). Since the levels of lymphocytes between B and T subgroups were different between the treatment patterns, we further explore the relation between dynamic change of the lymphocyte subgroups and treatment mode. As expected, dynamic changes of activated Tc cells related to treatment models, p = 0.038. Furthermore, the dynamics of activated Tc cells were also related to the evaluation(p = 0.043)(Supplementary Table).

Survival Analysis and Nomogram

The median follow-up time was 59.95 months, ranging from over 0 to 107 months. Overall survival time was discrepant in males and females(Log-rank test, p = 0.046). (Fig. 1B(a)). K-M survival analysis and log-rank test of intracranial germ cell tumors in neither different ages nor locations tended to be no statistical significance,(age, p = 0.778; locations, p = 0.243). Whereas, dynamic alternation of lymphocytes indicated that patients with increased Tc cells, activated T cells(including Th and Tc cells) were inclined to present significantly encouraging survival outcomes (p = 0.007, p = 0.021, and p = 0.028 respectively)(Fig. 1B(b),(c),(e)). Similarly, CD4⁺/CD8⁺ ratio within the normal range was related to a better prognosis, p = 0.016(Fig. 1B(d)).In comparison, T cells, Th cells, NK T cells, and B cells did not have any dictation on survival(p = 0.724, p = 0.765, p = 0.608, and p = 0.991,respectively). The combination of radiotherapy and chemotherapy had the best survival results, which is statistically significant(Fig. 1B(f),p < 0.001) Considering that the changes in T cells, Tc cells, B cells, and activated Tc cells were either statistical significance or associated with the survival outcomes of iGCTs patients, multivariate cox regression analysis was performed on these variables together with tumor indicators commonly used in clinical practice, serum AFP and HCG, tumor location and treatment methods as a panel to predict the prognosis(p < 0.001)(Table 3cox). It showed increased Tc cells was considered as an independent factor of better prognosis(p = 0.002,HR = 0.225 95%CI = 0.088—0.578). Interestingly, the increased CD3⁺ T cells were identified in the model as a factor related to poor prognosis, which is the opposite of the results of survival analysis. Taking into account that chemotherapy will significantly reduce the total number of leucocytes, and CD3⁺ T cells accounted for the largest proportion of lymphocytes, therefore, the independent prognostic significance of CD3 T cells remained obscure(p = 0.019, HR = 2.834 95%CI = 1.187—6.762).

To further visualize the prediction of the overall survival of iGCTs patients, a prognostic nomogram was established through Cox regression model analysis according to those significant indicators above (Tc cells, B cells, activated Tc, serum AFP and HCG, tumor location and treatment patterns)(Fig. 2A). Each factor in the nomogram was assigned a weighted number of points, and the sum of points for each patient was under a specific predicted 3- and 5-year OS. For internal validation, the bootstrapped calibration plot of the nomogram predicting 3- and 5-year OS performed well with the ideal model (Fig. 2B(a)(b)). The C-index of the multivariate prognostic model based on serum lymphocytes, tumor marker and sites of lesions 0.794 ± 0.069, and the integrated AUC curves as shown in Fig. 2B(b). The C-index of the proposed nomogram was 0.794, in comparison to those of tumor marker was 0.656 ± 0.312 and the integrated AUC curves were shown in Fig. 2B(b)(d).

Intracranial germ cell tumors (iGCTs) are a rare heterogeneous group of neoplasms mostly located in the pineal and/or sellar regions and mainly occur in children and adolescents^2,24. Teilum established the called “germ cell theory” believed that germ cell tumors emerged from primordial germ cells (PGCs) mis-migrated to the midline of the body, which led to CNS lesion occur²⁵. Confessedly, α-fetoprotein (AFP) and human chorionic gonadotropin (HCG) are credible tumor markers as confirmation of yolk sac tumors and choriocarcinomas^26,27 diagnosis and monitoring the response to treatments and follow-up, even lacking histological data.²⁷. Nevertheless, Although these two tumor indicators are fundamental in diagnosis and prediction of survival, it is challenging for patients with normal marker levels either in peripheral blood or in cerebrospinal fluid(CSF) to make clinical decisions and arduous to foresee the outcomes, if surgical resection cannot be performed. Increasing evidence supports that immune-related factors in the circulation have a major impact on treatment responses and clinical outcomes^28–30. Under the circumstances, this study was designed to study lymphocyte subsets among iGCTs patients and its related significance to prognosis.

The incidence of iGCTs is regarded as limited with discrepancy across North America, Europe and East Asia. The gender distribution(males = 120 cases, females = 25 cases, ratio:4.8) in our centre was consistent with the ratio of males largely exceeded females. Compared with it of 0.17 per million per year (gender distribution, males = 0.3, females = 0.07), the incidence rate has dropped to 0.1 per million per year(gender distribution, males = 0.13, females = 0.08), population composition ratio is analogous with data in the USA and Europe³¹. What's more, the cases of younger patients preceded the older in females(< 16 years old: 17 patients; >16 years old: 8 patients), while the younger and the older counted for half in males (< 16 years old: 60 patients; >16 patients: 60 patients).

Internationally, iGCT arises in the pineal (40–60%) and suprasellar (30–40%) regions or both locations as so-called bifocal (5–15%)³². The male to female ratio is 2–3:1 and in the pineal region up to 15:1³³, which being an extremely rare location in females. In our centre, the incidence of common sites is about 50%, and the ratio of males to females is 4:1. Similarly, the incidence of bifocal and rare other areas was also consistent with the international.

Cell-mediated immunity is regarded as a crucial soldier in anti-tumor response. Patients who suffered from brain tumors had 3-8-fold lower percentages of circulating lymphocytes compared to those of melanoma or breast cancer. That was observed in the study analyzed all of the brain cancer patients and is homogeneous with a recent study claiming that newly-diagnosed glioma has very low numbers of T cells in peripheral blood^34,35.

Brain tumor cells rupture the blood-brain barrier and they escape from tumor-associated antigens immune surveillance and finally lead to the entry of peripheral immune system components into the brain meanwhile communication between tumor microenvironment and peripheral circulation ^36,37. Our study confirmed that T cells, and Tc cells were significantly decreased, while activated T lymphocytes collected from serum were significantly increased. It was partially contradictory with the study of D.Kempuraj, etc. They found out CD4⁺T cells and CD⁺19 B cells increased after the treatment in both benign and malignant brain tumor patients, on the contrary, CD8⁺T cells decreased³⁸. The decreasing process of CD8⁺ T cells was commonly considered called “exhaustion.” CD8⁺ T cell exhaustion was first reported in a study using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection³⁹, which pointed under continuously stimulation from antigens, LCMV-specific CD8⁺ T cells exhibited restricted cell proliferation and impaired immune function, compared to conventional memory CD8⁺ T cells⁴⁰. These findings have also been confirmed in human patients with cancer^4142,43. Th lymphocytes bias promoted by serum exosomes and cytokines(eg. concentrations of colony-stimulating factors 2 and 3, as well as interleukin2, 4, and 13) was observed in peripheral blood in glioblastoma patients⁴⁴. It is consistent with the kinetics of lymphocytes in blood in our centre: patients with increased Tc(CD3⁺CD8⁺ ) cells were inclined to present significantly encouraging survival outcomes. Furthermore, the fact that increased CD8⁺ T cells in circulation play a crucial role in anti-tumor response is confirmed in other extracranial tumors. Interestingly, NK cells did not present any significancy change or relation in either course of chemotherapy or prognosis in our results. Commonly, they are responsible to cancer immune surveillance and killing by natural cytotoxicity triggered rapidly upon stimulation through germline-encoded cell surface receptors. We assumed it is similar to the fact that glioblastoma tumor cells and tumor microenvironment suppressed natural killer(NK) cells via expression of factors such as transforming growth factor (TGF)-β,and impairs NK cells by downregulating the mTOR pathway. B cells(CD19⁺) induced humoral immunity in patients with brain tumors was less markedly affected. Similarly, either increasing or non-increasing B(CD19⁺) cells monitored in this study cannot indicate any outcomes. Survival analysis confirmed that increased CD3⁺T cells are related to improved lifetime. Contradictorily, increased CD3⁺T cells indicated increased death risk in multivariate cox regression analysis. Thus we take the evidence that chemotherapy leads to reduction of leukocytes significantly(confirmed both in our center and common sense, p = 0.000) and CD3⁺ T cells count for the largest into consideration, which might lead to the confusing result that patients with increased CD3⁺T cells might not have the potential to enjoy life more.

Nonetheless, there are limitations of our study that should be claimed, and results should be interpreted with prudence. Firstly, considering the lack of a larger sample, some of the results could not be identified as significant. We would like to expand the scale of patients participants in future research. Besides, we did not compare the long-term dynamics of lymphocyte subsets, which are currently in progress under surveillance in another prospective program.

In conclusion, lymphocyte subsets distinct after chemotherapy on diagnosed intracranial germ cell tumors patients. Except for elevation of activated cytotoxic T cells, T lymphocytes, Tc cells, and B cells decreased after first chemotherapy, and dynamics of Tc cells, activated T cells were closely associated with exceptional prognosis in iGCTs patients and might be a potential auxiliary prognostic index.

iGCTs: intracranial germ cell tumors

NGGCTs:non-germinoma germ cell tumors

GCTs: germ cell tumors

CSF: cerebrospinal fluid

AFP: α-fetoprotein

HCG: human chorionic gonadotropin

HIV: human immunodeficiency virus

NSCLC: non-small cell lung carcinoma

E: etoposide

P: cisplatin

B: bleomycin

C: carboplatin

I: ifosfamide

WBNT: whole-brain radiotherapy

CSI: craniospinal irradiation

GTV: gross target volume

CTV: clinical target volume

TMZ: temozolomide

OS: Overall survival

TTP:time to progression

PGCs: primordial germ cells

ES: embryonal stem

NSC: neural stem cells

Th cells: T helper cells

Tc cells: T toxic

Treg cells:T regulatory cells

TNF: tumor necrosis factor

IL: Interleukin

CTL/Tc cells: cytotoxic T cells

LCMV: lymphocytic choriomeningitis virus

TCR: T cell receptor

CAR-T: chimeric antigen receptor T cell

Bif: Bifocus of pineal gland and saddle

Mul: Multiple tumors

S-com: Single at pineal gland/basal ganglia/saddle;

S-uncom: Single tumor at others sites

Ventricles: Violation of ventricle systems

Inc: Group of Increased lymphocytes

Non-Inc: Group of Non-increased lymphocytes

Author contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hongyu Chen, Jiangen Chen, Xiaopin Lin and Hairong Wang. The first draft of the manuscript was written by Hairong Wang and He Huang, Zhongpin Chen, Yonggao Mou, Peidong Chi, Qunying Yang, and Chengcheng Guo critically revised the manuscript. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Hairong Wang*: Collected the data and wrote this manuscript

He Huang*: Revising the work critically for important intellectual content.

Yonggao Mou, Peidong Chi: Interpretation for partial important intellectual content

Xiaopin Lin,: Collect the data and analysis.

Zhongpin Chen#, Qunying Yang#, Chengcheng Guo#(main corresponding author): Substantial conception interpretation and design of the work.

*These authors contributed equally to this work.

# Corresponding author.

Competing interests: The authors have no relevant financial or non-financial interests to disclose

FUNDING

This research was supported by the Basic and Applied Basic Research Project of Guangdong (2019A1515010702) and the Science and technology planning project of Guangzhou (202002030114)

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Table 1, 2 are available in the Supplemental Files section.

Table 3 Multivariate cox regression analysis serum lymphocyte subsets together with tumor indicators(serum AFP and HCG), tumor location and treatment methods. * represents significant indicators

Cox Regression of indicators in Nomogram before treatment
	B	SE	Wald	df	Sig	Exp(B)	95.0% Exp(B) CI
	B	SE	Wald	df	Sig	Exp(B)	lower limit	upper limit
GroupCD3	1.042	0.444	5.509	1	0.019*	2.834	1.187	6.762
GroupCD3CD8	-1.491	0.480	9.625	1	0.002*	0.225	0.088	0.578
GroupCD19	0.144	0.395	0.133	1	0.715	1.155	0.532	2.508
GroupCD4CD25	-0.154	0.375	0.170	1	0.680	0.857	0.411	1.786
GroupCD8CD25	-0.646	0.379	2.905	1	0.088	0.524	0.249	1.102
AFP	-1.501	0.372	16.315	1	<0.001*	0.223	0.108	0.462
HCG	0.589	0.336	3.071	1	0.080	1.802	0.933	3.483
Sites(Bif)			2.553	4	0.635
Sites(Mul)	-0.576	0.738	0.610	1	0.435	0.562	0.132	2.386
Sites(S-com)	-0.584	0.540	1.166	1	0.280	0.558	0.193	1.609
Sites(S-uncom)	0.115	0.804	0.021	1	0.886	1.122	0.232	5.422
Sites(Ventrical)	-0.282	0.573	0.242	1	0.623	0.754	0.245	2.320
Treatment(Sugery-radio-chemo)			25.428	2	<0.001*
Treatment(Chemo)	1.067	0.745	2.051	1	0.152	2.906	0.675	12.511
Treatment(Radio-chemo)	-1.274	0.621	4.208	1	0.04*	0.280	0.083	0.945

No competing interests reported.

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Integrated Analysis of Serum Lymphocytes Develops a Nomogram for Predicting Prognosis of Intracranial Germ Cell Tumors to Chemo-Radiotherapy

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Comparison of Changes in Lymphocyte Subsets

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