Serum Histone Succinylation: A Predictive Biomarker for Malignant Solid Tumors
Conditions: Malignant Tumors
Sponsor: Yinghua Ji

Clinical Study Protocol
Project Title:
Predictive Value of Serum Histone Succinylation in Malignant Solid Tumors
Leading institutions:
The First Affiliated Hospital of Xinxiang Medical University
School of Life Sciences, East China Normal University
Version: V4.1
Date: 19-Jul-2025
Principal Investigators: Ping Lu, Jiemin Weng
Protocol Approval：
Compliance Statement
Participants must comply with the Declaration of Helsinki and commit to conducting this study in
accordance with this protocol. They must be trained and the study can only be carried out after
obtaining written approval from the ethics committee and written informed consent from the
subjects. Any revision to the protocol requires re-approval.

一、scenario summary
In recent years, with the in-depth research on post-translational modifications of
proteins, scientists have discovered that histone succinylation modification, as a novel
protein modification method, is closely related to the occurrence, development and
prognosis of tumors. Succinylation modification affects processes such as cell
metabolism, proliferation and apoptosis by altering the physicochemical properties
and functions of proteins. In tumor cells, the abnormal expression of succinylation
modification is closely related to tumor type, stage and prognosis, providing new
ideas for the early diagnosis of tumors. This project uses blood serum samples and
detects and analyzes the level of histone succinylation modification in the serum
through specific histone succinylation modification antibodies. Combined with other
clinical information, it aims to achieve early, rapid and accurate diagnosis of
pan-tumors, realize the rapid screening of tumors with "one tube of blood", and
promote the transformation of precise early diagnosis of tumors from "gene-driven" to
"epigenetic metabolism-driven". The implementation of this project aims to explore
the feasibility of histone succinylation modification in serum as a tumor marker,
providing more effective biomarkers for tumor early screening, therapeutic effect
evaluation and prognosis. This will help develop more accurate and convenient blood
testing methods in the future, benefiting a wider patient population and having
significant social value.
二、introduction
2.1 Research background, project objectives and the foundation of previous research
2.1.1 Research background
Cancer has become a major social, economic and public health issue in the 21st
century and is one of the leading causes of death worldwide. In 2020, both the number
of new cancer cases and deaths in China ranked first in the world. With the rapid
development of medical technology, early diagnosis of tumors is of vital importance
for improving the survival rate and quality of life of patients. However, existing tumor
diagnostic methods, such as imaging examinations, histopathological examinations,
and traditional tumor marker detection, still have deficiencies in terms of sensitivity,

specificity, and early detection capabilities. They cannot achieve the detection of
multiple tumors with a single method, and may have limitations such as high
professional requirements, long time consumption, and high detection costs.
Therefore, it is of vital importance to develop a novel pan-tumor screening and
diagnosis method that is low-cost, highly sensitive and highly specific.
Free nucleosomes in cells are nucleosomes released into circulating tissues after
apoptosis or necrosis of cells, and they retain some specific information of the origin
cells and tissues. The histone modification status on the free nucleosomes produced
by tumor cells reflects the metabolic state and epigenetic changes of tumor cells.
Therefore, histone modification of free nucleosomes in cells is expected to become a
new biomarker for non-invasive tumor detection. Although previous studies have
found the association between histone succinylation modification and tumors, and
free nucleosomes in cells retain specific histone modifications of the origin tumor
cells, histone succinylation modification of free nucleosomes in cells has not yet been
applied to tumor screening and detection. Therefore, it is urgently necessary to study
whether histone succinylation modification of free nucleosomes can be used as a
low-cost, highly sensitive and highly specific pan-tumor marker and applied in the
early screening, therapeutic effect evaluation and prognosis of tumors.
Succinylation modification, as a widely existing post-translational modification of
proteins, has potential biomarker value in malignant solid tumors. Besides tumors,
existing studies have shown that some diseases (such as inflammatory diseases,
infectious diseases, metabolic diseases, cardiovascular diseases, neuro-psychological
diseases, etc.) have certain correlations with non-histone succinylation modification.
The role of histone succinylation modification in other diseases is currently lacking in
relevant clinical research evidence and awaits further exploration in this study.
Therefore, this research focuses on malignant solid tumors, not only filling the
knowledge gap in this field but also laying the foundation for future expansion to
other diseases.
2.1.2 project objective
The core objective of the project is to systematically verify and establish the clinical

application value of serum histone succinylation modification as a novel, efficient and
non-invasive pan-tumor biomarker.
Objective 1: Verify the potential of pan-tumor screening: Through large-scale
case-control studies, it is clearly confirmed that there are significant differences in
histone succinylation levels in serum between patients with malignant solid tumors
and healthy individuals, thereby providing a solid evidence-based basis for
developing it into a blood-based early screening tool suitable for multiple tumor
types.
Objective 2: Exploring clinical application scenarios: The project will conduct an
in-depth analysis of the association between serum histone succinylation levels and
different types of tumors as well as different tumor stages, and evaluate its ability to
distinguish tumor types and reflect disease progression. Meanwhile, analyze its
correlation with therapeutic effects (changes in levels before and after surgery,
radiotherapy, and systemic treatment) and clinicopathological characteristics, and
explore its application prospects in dynamic monitoring of therapeutic effects and
prognosis assessment.
2.1.3 Preliminary research foundation
（1）Professor Weng Jieming's research group from East China Normal University
previously discovered that histone succinylation modification was significantly higher
in various solid tumors than in adjacent tissues. The results are shown in the following
figure: Histone succinylation modification at the H4K31 site was significantly higher
in various solid tumors than in adjacent tissues.
Professor Weng Jieming's research group previously prepared histones from human
breast cancer, prostate cancer, lung cancer, kidney cancer, and lung cancer tissue
samples from mice. The Western blot results (see Figure 1) indicated that the levels of
succinylation modifications at histone H4K31 and H3K14 sites in cancer tissues were
significantly higher than those in adjacent tissues, such as breast cancer (Figure 1A),
prostate cancer (Figure 1B), renal cancer (Figure 1C), and lung cancer (Figures 1D
and 1E). It reveals that histone succinylation modification can serve as a potential
biomarker for tumorigenesis and development.

Figure 1: The succinylation modification at the histone H4K31 site in tumor tissues
was significantly higher than that in adjacent tissues: (A) Western blot analysis of the
levels of succinylation modification and histone acetylation modification at the
histone H4K31 site in human breast tumor samples compared with those in adjacent
tissues; (B) Western blot was used to analyze the levels of succinylation modification
and histone acetylation modification at the histone H4K31 site in human prostate
tumor samples compared with those in adjacent tissues. (C) Western blot was used to
analyze the levels of succinylation modification and histone acetylation modification
at the histone H4K1 site in human renal tumor samples compared with those in
adjacent tissues. (D) Western blot was used to analyze the levels of succinylation
modification and histone acetylation modification at the histone H4K31 site in human
lung tumor samples compared with those in adjacent tissues.
Based on the findings in solid tumors, the level of histone succinylation modification
in tumor tissues was significantly higher than that in adjacent tissues. Therefore, the
research group, in collaboration with hospitals, collected blood samples from patients
with different types of tumors and found that the succinylation modification at the
H3K23 site of free nucleosome histones in the blood of tumor patients was
significantly higher than that of normal people. However, the number of serum

samples from cancer patients still needs to be further expanded.
The research group previously isolated free nucleosomes from human colon cancer,
gastric cancer, breast cancer, lung cancer, pancreatic cancer, multiple myeloma, liver
cancer,
cervical
cancer,
prostate
cancer,
ovarian
cancer,
esophageal
cancer,
cholangiocarcinoma and normal people's blood for Western blot. The results and
gray-scale
analysis
statistics
(see
Figure
2)
indicate
that
the
succinylation
modification of free nucleosome histone H3K23 sites in the blood of patients with
different types of tumors (for specific information on different tumor types, see Figure
3) is significantly higher than that of normal people, revealing that the succinylation
modification of free nucleosome histone in the blood of tumor patients may serve as a
biomarker for the early diagnosis of tumors. However, at present, due to the small
sample size, it is necessary to carry out this project to further expand the sample size
and conduct in-depth research.

Figure 2 shows that the levels of free nucleosome histone succinylation modification
in the blood of patients with different types of tumors are significantly higher than
those of normal people: Enrich the free nucleosomes in the blood of tumor patients.
Use histone H3K23 specific site succinylation modification and histone H3
acetylation modification antibodies to analyze the levels of histone succinylation
modification and acetylation modification of free nucleosomes in the blood of
different types of tumor patients by Western blot. And conduct gray-scale analysis and
statistics on the Western blot results. Among them, N: normal human serum; T: Serum
of cancer patients. Specific information on different types of tumors is shown in
Figure 3.
Blood samples from different human tumors

Figure 3. Information of different types of tumor samples and information of normal people
2.2
Risk/Benefit assessment
risk assessment：
This study was an observational one, and the core intervention was only a single
venous blood sample collection of 3mL (approximately 1ml of serum). This operation

is a routine and mature medical practice in clinical practice, with extremely low and
controllable risks.
Benefit evaluation：
Cancer patients: They will not receive immediate individualized diagnosis or
treatment recommendations from this study. However, the data it contributes will
drive the development of better tumor markers, which may benefit its future disease
management (such as more precise follow-up monitoring).
Healthy control subjects: Contribute to establishing baseline standard values for
healthy populations and promote public health. After the research is completed and
the method is mature, the baseline data in which it participated may provide a
reference background for its own future similar screening.
三、Research objectives and endpoints
3.1 objective
3.1.1 main purpose
To evaluate the differences in serum histone succinylation between malignant solid
tumors and healthy individuals, in order to verify its early screening value.
3.1.2 secondary objective
To clarify the correlation between histone succinylation modification in serum and
different types of tumors and tumor stages, and to statistically analyze the correlation
between histone succinylation in serum and therapeutic effects (including surgical
treatment, radiotherapy, and systemic therapy) as well as clinicopathology.
3.2 evaluation index
The evaluation indicators are: the sensitivity, specificity, positive predictive value,
negative predictive value of the serum histone succinylation screening method, and
the predictive value of the Youden index (early diagnosis, therapeutic effect
evaluation and prognosis).
四、study population
4.1
inclusion criteria
case group

1）≥18 years old, gender not limited;
2 ）Malignant solid tumors diagnosed by cytology or histology require subsequent
anti-tumor
treatment
(systemic
therapy,
radical
concurrent
chemoradiotherapy/radiotherapy, or surgical treatment).
control group
1) ≥18 years old, gender not limited;
2) Non-tumor patients undergoing health check-ups;
4.2 exclusion criteria
1) Pregnant women 2) Those with severe mental disorders or language communication
disorders; 3) Other circumstances where the researcher deems it unsuitable to participate
in this study.
The control group was the same as the case group.
五、study design
5.1 overall design
This study is a single-center prospective case-control study.
5.2
Research design process
5.2.1 Study the specific implementation process
1. Phase One: Screening and enrolling the population
i. Conduct ethical reviews and clinical research protocol reviews for the project;
ii. It is planned to select 500 patients with malignant solid tumors, collect their basic
information and sign the "Informed Consent Form".
iii: It is planned to select 500 healthy subjects and collect basic information.
2. Phase Two: Collect blood samples for serum histone succinylation detection
Blood samples were collected from all 500 enrolled individuals and 500 healthy
subjects who met the requirements for serum histone succinylation detection.
3. Phase Three: Statistical analysis of Screening Results
i. Western blot was used to evaluate the differences in serum histone succinylation
between malignant solid tumors and healthy individuals to verify its early screening
value.

ii. Statistically analyze the correlation between serum histone succinylation and
tumor efficacy (including surgical treatment, radiotherapy, and systemic therapy) as
well as clinicopathology. The therapeutic effect was evaluated using RECIST1.1
criteria.
5.2.2 Research flowchart

5.2.3 Blood collection process table
grouping
Screening
period
healthy person
D-7—D0
Cancer patients are
receiving systemic
treatment
Chemotherapy cycle
C1D1
C2
C3
C4
C5
C6
D-7—D0
D1±
3
D1±3
Cancer patients who
receive radical
chemoradiotherapy/radioth
erapy
D-7—D0
After the completion of
radiotherapy/chemoradiotherapy
Four to six weeks after the
completion of
radiotherapy/chemoradiother
apy
DendDend+3
On the day of follow-up
A tumor patient
undergoing surgery
D-7—D0
postoperation
Postoperative discharge day/the
day before discharge
D1—D3
5.3 statistic analysis
This study adopted a mismatched case-control study (500 cases in the case group and
500 cases in the healthy control group). The sample size was determined based on the
effect size of the previous pre-experiment (Cohen's d=1.2) and considering a 10%
dropout rate. All statistical analyses were performed using R, with a test level of
α=0.05. Main analysis 1) Comparison of histone succinylation levels between groups
- After Shapiro-Wilk normality and Levene homogeneity of variance tests,
independent sample t-tests (conforming to normal distribution) or Mann-Whitney U
tests (non-normal data) were selected. Report mean difference (MD)/median
difference and 95% confidence interval (CI), Cohen's d effect size; 2) Diagnostic

efficacy evaluation - Calculate the area under the curve (AUC) and its Bootstrap 95%
CI through the ROC curve, and determine the optimal cut-off value by maximizing
the Youden index. And calculate the sensitivity, specificity, positive predictive value
(PPV), negative predictive value (NPV), and their exact Clopper-Pearson 95% CI.
Secondary analysis: 1) Subgroup correlation - One-way analysis of variance (ANOVA)
or Kruskal-Wallis test was used to compare the level differences among different
tumor types (Bonferroni-adjusted post hoc test), and Cuzick's trend test was used to
analyze the ordered association of TNM stages; 2) Therapeutic effect evaluation - The
level changes before and after treatment were evaluated using paired t-test /Wilcoxon
signed-rank test; 3) Prognostic value - The baseline succinylation level (median
grouping) was included in the Cox proportional hazards model. After adjusting for
age, stage, and treatment regimen, the hazard ratio (HR) and 95% confidence interval
(CI) were calculated. The survival curve was plotted using the Kaplan-Meier method
(Log-rank test).Multivariate analysis: A Logistic regression model was constructed
(including succinylation level and covariates such as age and gender). AUC was
compared by DeLong test, goodness-of-fit was evaluated by Hosmer-Lemeshow
calibration curve, and clinical net benefit was analyzed by decision curve (DCA). If
multi-site modifications are detected, LASSO regression is used to screen biomarker
combinations.
Quality
control:
Grubbs
test
/Winsorize for
outliers,
multiple
imputation (MICE) for filling <5% of missing covariates. Sensitivity analysis:
Robustness was verified by excluding stage I tumors, and the 70%-30% dataset was
split for cutoff value training - validation. All multiple comparisons were controlled
for the false discovery rate (FDR) using the Benjamini-Hochberg method.
六、Ethical requirements
Clinical research will comply with relevant regulations such as the Declaration of
Helsinki of the World Medical Congress. The research can only be implemented after
it has been approved by the ethics committee before it commences.
Before each subject is enrolled in this study, the researcher is responsible for
providing the subject or their agent with a complete and comprehensive introduction

to the purpose, procedures and possible risks of this study, and informing the subjects
that they can refuse to participate or withdraw from this study at any stage without
discrimination or retaliation, and that their medical treatment and rights will not be
affected. After fully understanding and agreeing, the subjects signed the written
informed consent form, which was retained as a clinical research document for future
reference. During the research process, researchers will protect the personal privacy
and data confidentiality of the subjects. In this study, the informed consent form was
electronically signed. The doctor/working group coordinator communicated with the
subjects on-site. Before the subjects were enrolled in the study, the doctor would
inquire and record the subjects' medical history. If the subject is a qualified inclusion
and voluntarily participates, they will be asked to sign an informed consent form. All
electronic informed consent forms will be filed.

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