Serum Histone Succinylation as a Novel Prognostic Biomarker in Hematological Malignancies
Conditions: Leukemia, Lymphoma
Sponsor: The First Affiliated Hospital of Xinxiang Medical College

Clinical research plan
Project Title: Predictive Value of Serum Histone Succinylation
in Hematological Cancers
Group leader unit: The First Affiliated Hospital of Xinxiang Medical
College
School of Life Sciences, East China Normal University
Version number: V 4.0
Version Date : 2025.08.12​
​
Project Leader: Lu Ping and Weng Jiemin
Declaration of Conformity
Abide by the Declaration of Helsinki and commit to conducting this study in accordance with this
protocol. Participants must be trained. The study will be conducted after obtaining written approval
from the ethics committee and written informed consent from the subjects. Any amendments to the
protocol require re-approval.

I. Summary of the Plan
In recent years, important breakthroughs have been made in the field of protein
post-translational
modification
research.
Among
them,
histone
succinylation
modification, as a new metabolic-related epigenetic regulatory mechanism, plays a
key role in the occurrence and development of tumors. Studies have shown that
succinylation modification significantly affects the dynamic structure of chromatin
and the gene transcription regulatory network by changing the charge characteristics
and spatial conformation of histones, thereby participating in malignant biological
behaviors such as metabolic reprogramming, abnormal proliferation and apoptosis
escape of tumor cells. In particular, in hematological malignancies (such as leukemia,
lymphoma, multiple myeloma, etc.), the abnormal expression pattern of histone
succinylation modification is significantly correlated with disease subtype, clinical
stage and treatment response, which provides an important theoretical basis for the
development of new molecular markers for hematological tumors. This project
focuses on hematological malignancies and innovatively uses patient peripheral
blood serum as the research object. It uses highly specific histone succinylation
modification antibodies combined with high-sensitivity mass spectrometry detection
technology to establish a precise quantitative detection system for serum histone
succinylation
modification.
By
systematically
analyzing
the
characteristic
succinylation profiles of different hematologic malignancies and at various stages of
disease progression, combined with multidimensional clinical data integration, the
goal is to construct a multi-parameter prediction model based on serum histone
succinylation profiles for early diagnosis, therapeutic efficacy assessment, and
prognosis prediction of hematologic malignancies. This research has the potential to
overcome existing technical bottlenecks, enabling a technological revolution in the
non-invasive, single-tube blood test for hematologic malignancies and promoting a

shift in the diagnostic paradigm from traditional "gene-driven" to innovative
"epigenetic metabolism-driven" approaches. This project will be the first to
systematically evaluate the translational application of serum histone succinylation in
the clinical diagnosis of hematologic malignancies. It will not only provide new
scientific insights into the epigenetic metabolism regulation mechanisms of
hematologic malignancies, but also potentially offer a novel, efficient, minimally
invasive strategy for early screening and dynamic monitoring of hematologic
malignancies in clinical practice. This project holds significant theoretical significance
and promising clinical applications. The findings will open new avenues for the
precision diagnosis and treatment of hematologic malignancies and provide valuable
insights for epigenetic marker research in other systemic malignancies.
II. Introduction
2.1 Research background , project objectives and preliminary research basis
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, China ranked
first in the world in both new cancer cases and deaths . With the rapid development
of medical science and technology, early diagnosis of tumors is crucial to improving
patients' survival rates and quality of life . However, existing tumor diagnostic
methods, such as imaging examinations, histopathological examinations and
traditional tumor marker tests, still have shortcomings in sensitivity, specificity and
early detection capabilities . They cannot detect multiple tumors in one method , and
may have limitations such as high professional requirements, long time consumption
and high testing costs. Therefore, it is crucial to develop a new pan-tumor screening
and diagnostic method with low cost, high sensitivity and high specificity .
Cell-free nucleosomes are nucleosomes released into circulating tissues after
apoptosis or necrosis, retaining some specific information about their cell and tissue
of origin. The histone modification status on cell-free nucleosomes produced by
tumor cells reflects the metabolic state and epigenetic changes of tumor cells.

Therefore, histone modifications on cell-free nucleosomes are expected to become
new biomarkers for non-invasive tumor detection. Although studies have found an
association
between
histone
succinylation
and
tumors,
and
that
cell-free
nucleosomes retain histone modifications specific to the tumor cell of origin, histone
succinylation on cell-free nucleosomes has not yet been applied to tumor screening
and detection. Therefore, it is urgent to study whether histone succinylation on
cell-free nucleosomes can be used as a low-cost, highly sensitive, and highly specific
pan- tumor marker for early cancer screening, efficacy assessment, and prognosis.
2.1.2 Project Objectives
The core goal 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: Validate the potential for pan-tumor screening: Through a
large-scale case-control study, clearly confirm that serum histone succinylation levels
are significantly different between patients with malignant hematological tumors and
healthy people, thereby providing a solid evidence-based basis for developing it as a
blood-based early screening tool applicable to multiple tumor types.
Objective 2: Exploring Clinical Applications : This project will conduct in-depth
analysis of the association between serum histone succinylation levels and different
tumor types and stages, evaluating their ability to distinguish tumor types and reflect
disease progression. Furthermore, the project will analyze their correlation with
treatment efficacy (e.g., changes in levels before and after surgery, radiotherapy, and
systemic treatment) and clinical pathological features, exploring their potential
applications in dynamic efficacy monitoring and prognostic assessment.
2.1.3 Preliminary Research Foundation
(1) Professor Weng Jiemin's research group at East China Normal University
previously found that histone succinylation modification was significantly higher in
various solid tumors than in adjacent tissues. The results are shown in the figure
below: Histone H4K31 succinylation modification was significantly higher in various
solid tumors than in adjacent tissues.

Professor Weng Jiemin's research group previously prepared histones from
human breast, prostate, lung, and kidney cancer tissue samples, as well as mouse
lung cancer tissue samples. Western blot results (Figure 1) showed that succinylation
of histones at H4K31 and H3K14 sites was significantly elevated in cancer tissues
compared to adjacent adjacent tissues, as seen in breast cancer (Figure 1A), prostate
cancer (Figure 1B), kidney cancer (Figure 1C), and lung cancer (Figures 1D and 1E).
This suggests that histone succinylation could serve as a potential biomarker for
tumor development and progression.
Figure 1. The succinylation modification of histone H4K31 site was significantly higher in tumor
tissues than in adjacent tissues: (A) Western blot analysis of the levels of succinylation
modification and histone acetylation modification of histone H4K31 site in human breast tumor
samples compared with adjacent tissues; (B) Western blot analysis of the levels of succinylation
modification and histone acetylation modification of histone H4K31 site in human prostate tumor
samples compared with adjacent tissues; (C) Western blot analysis of the levels of succinylation
modification and histone acetylation modification of histone H4K31 site in human kidney tumor
samples compared with adjacent tissues; (D) Western blot analysis of the levels of succinylation
modification and histone acetylation modification of histone H4K31 site in human lung tumor
samples compared with adjacent tissues; (E) Western Blot analysis of the levels of histone H4K31
succinylation and histone acetylation in mouse lung tumor samples compared with normal tissues;
N: adjacent tissue to the tumor; T: tumor tissue.
(2) Based on the findings in solid tumors, the level of histone succinylation
modification in tumor tissue is significantly higher than that in adjacent tissues.
human breast tumor
human prostate tumor
human lung tumor
human kidney tumor
mouse lung tumor
N
T
T

Therefore, the research team cooperated with the hospital to collect blood samples
from patients with different types of tumors. It was found that the succinylation
modification of the free nucleosome histone H3K23 site in the blood of tumor patients
was significantly higher than that of normal people. However, the number of serum
samples from tumor patients needs to be further expanded.
2.2 Risk/Benefit Assessment
Risk Assessment:
This study was observational, and the core intervention consisted of a single
venous blood sample collection (approximately 1 mL). This procedure is a routine
and established clinical practice with extremely low and manageable risks .
Benefit evaluation:
Cancer patients will not receive immediate personalized diagnosis or treatment
recommendations from this study. However, the data they contribute will promote
the development of better tumor markers and may benefit their future disease
management (such as more accurate follow-up monitoring).
Healthy controls: Contribute to establishing baseline values for healthy people
and promote public health. Once the study is completed and the methods are
mature, the baseline data from their participation may provide a reference
background for future similar screening.
3. Study Objectives and Endpoints
3.1 Purpose
3.1.1 Primary Objective
To evaluate the differences in serum histone succinylation levels between
patients with hematologic malignancies and healthy individuals, and to confirm
whether there is a significant difference in serum histone succinylation levels
between these two groups.
3.1.2 Secondary Objectives
To determine the correlation between serum histone succinylation modification
levels and different types of tumors as well as tumor stages, and to statistically

analyze the relationship between serum histone succinylation and therapeutic
outcomes (including chemotherapy, hematopoietic stem cell transplantation, and
cellular immunotherapy) as well as clinical pathological features.
3.2 Evaluation Metrics
The evaluation metrics include: sensitivity, specificity, positive predictive value,
negative predictive value, and Youden’s index of the screening method for serum
histone succinylation modification levels, assessing their predictive value for early
diagnosis, treatment efficacy, and prognosis.
4. Study Population
4.1 Inclusion Criteria
Case group
1) ≥18 years old , regardless of gender;
2) Hematological malignancies confirmed by cytology, histology , or molecular
biology (such as leukemia, lymphoma, multiple myeloma, etc.), who require
subsequent anti-tumor treatment ( chemotherapy , hematopoietic stem cell
transplantation, radiotherapy, or cellular immunotherapy ) ;
control group
1) ≥18 years old , regardless of gender;
2) Non-cancer patients undergoing health checkups;
4.2 Exclusion criteria
1) Pregnant women
2) Those with severe mental disorders or language communication barriers
3) Other circumstances that the researcher judges to be unsuitable for participation in
this study
The control group was the same as the case group
V. Research Design
5.1 Overall Design

This study was a single-center prospective case-control study.
5.2 Research Design Process
5.2.1 Study the specific implementation process
1. Phase 1: Screening population for enrollment
i. Conduct ethical review and clinical research plan review for the project;
ii. 200 patients with hematological malignancies will be selected , basic
information collected , and informed consent signed ;
iii: 200 healthy subjects are planned to be selected and basic information
collected;
2.
Second
stage:
Collect
blood
samples
and
perform
serum
histone
succinylation test
Blood samples were collected from all 200 eligible participants and 200
healthy subjects for serum histone succinylation testing ;
3. The third stage: statistical analysis of screening results
i. Evaluate the differences in serum histone succinylation between patients with
hematological malignancies and healthy controls to validate its value in early
screening.
ii. Statistical analysis of the correlation between serum histone succinylation and
tumor efficacy (including chemotherapy , hematopoietic stem cell transplantation,
cellular immunotherapy ) and clinical outcomes . ( Efficacy evaluation was
performed using RECIST 1.1 criteria )
5.3 Statistical analysis
This study employed an unmatched case-control study (500 cases in the case
group and 500 healthy controls). The sample size was determined based on a
pre-experimental effect size (Cohen's d = 1.2) and a 10% dropout rate. All statistical
analyses were performed using R , with an α level of 0.05. The following primary
analyses were performed: 1) Comparisons of histone succinylation levels between
groups were performed using the independent sample t-test (for normal
distribution) or the Mann-Whitney U test (for non-normal data) , after

Shapiro-Wilk normality and Levene's homogeneity of variance tests . Mean
differences (MD)/median differences with 95% confidence intervals (CIs) and
Cohen's d effect sizes were reported. 2) Diagnostic performance was evaluated by
calculating the area under the receiver operating characteristic (ROC) curve (AUC)
and its bootstrap 95% CI. The optimal cutoff value was determined by maximizing
the Youden index. Sensitivity, specificity, positive predictive value (PPV), negative
predictive value (NPV), and their exact Clopper-Pearson 95% CIs were also
calculated. Secondary analyses included : 1) Subgroup associations: One-way
analysis of variance (ANOVA) or the Kruskal-Wallis test (Bonferroni correction post
hoc test) was used to compare differences in levels between different tumor types, and
Cuzick's trend test was used to analyze ordered associations with TNM stage. 2)
Efficacy assessment: Changes in levels before and after treatment were evaluated
using paired t-tests/Wilcoxon signed-rank tests. 3) Prognostic value: Baseline
succinylation levels (grouped by median) were included in a Cox proportional hazards
model. Hazard ratios (HRs) and 95% CIs were calculated after adjusting for age, stage,
and treatment regimen. Survival curves were plotted using the Kaplan-Meier method
(log-rank test). Multivariate analysis : A logistic regression model was constructed
(incorporating succinylation levels and covariates such as age and sex). The DeLong
test was used to compare area under the curve (AUC). Goodness of fit was assessed
using the Hosmer-Lemeshow calibration curve. Net clinical benefit was analyzed
using a decision curve analysis (DCA). If multiple marker modifications were
detected, LASSO regression was used to select marker combinations. Quality control
included Grubbs' test/Winsorize to address outliers, and multiple imputation with ice
cream (MICE) to impute missing covariates <5%. Sensitivity analyses included
excluding stage I tumors to validate robustness, and using a 70%-30% dataset split for
training-validation cutoffs. All multiple comparisons were performed using the
Benjamini-Hochberg method to control the false discovery rate (FDR).
VI . Ethical Requirements
Clinical research will adhere to relevant regulations such as the World Medical

Association's Declaration of Helsinki. Prior to commencement, the study must be
approved by the ethics committee.
Before each subject is selected for this study, the researcher is responsible for
fully and comprehensively introducing the purpose, procedures and possible risks of
the study to the subject or his/her agent, and letting the subject know that they can
refuse to participate or withdraw from the study at any time at any stage of the study
without being discriminated against or retaliated, and that their medical treatment
and rights will not be affected. After the subject fully understands and agrees, he/she
will sign a written informed consent form, which will be retained as a clinical
research document for future reference. During the study, the researcher will protect
the subject's personal privacy and data confidentiality. This study uses electronic
signatures for informed consent, and doctors/working group coordinators will
communicate with the subjects on-site. Before the subject is selected for the study,
the doctor will inquire about and record the subject's medical history. If the subject is
an eligible participant and voluntarily participates, the subject will be asked to sign
the informed consent form. All electronic informed consent forms will be archived.

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