Clinical Research on Efficacy and Safety of Combined Chemotherapy With Arsenic Trioxide in Stage 4/M Neuroblastoma：A Prospective，Single-arm, Open-label, Multi-center Study
Phase: PHASE2
Conditions: Neuroblastoma
Sponsor: Yang Li

Phase II Study of Combined Chemotherapy
With Arsenic Trioxide in Stage 4/M
Neuroblastoma
(NCT03503864)
Protocol
Version:2.0
March 17 2020

CONTENTS
Phase II Study of Combined Chemotherapy With Arsenic Trioxide in
Stage 4/M Neuroblastoma (NCT03503864)............................................1
1. Background.............................................................................................................. 3
2. STUDY OBJECTIVES............................................................................................7
2.1 PRIMARY OBJECTIVE..................................................................................7
2.2 SECONDARY OBJECTIVES......................................................................... 8
3. STUDY DESIGN......................................................................................................8
3.1. DESCRIPTION OF STUDY...........................................................................8
3.2. SELECTION OF STUDY POPULATION.....................................................9
3.4. Pre-treatment Examination............................................................................ 11
3.5. Treatment Protocol........................................................................................ 12
3.6. Adverse Reactions and Management.............................................................15
3.7. Observation Indicators and Follow-up.......................................................... 17
3.8. Criteria for Efficacy Evaluation.....................................................................18
3.9. Safety Evaluation Criteria..............................................................................20
3.10. Statistical Analysis Methods........................................................................20
4. Reference................................................................................................................ 20

1. Background
Neuroblastoma (NB) is the most common extra‐cranial solid tumor in children,
originating from primitive neuroblasts and can develop in any region of the
sympathetic nervous system. It accounts for 8%-10% of the incidence of childhood
malignancies and contributes to 15% of cancer‐related mortality in children. Children
with high-risk NB (HR-NB) exhibit a high degree of malignancy and are
characterized by an aggressive progression, a heightened probability of early distant
metastasis, and a significantly increased risk of mortality [1-2]. The 5-year survival rate
for stage 4/M neuroblastoma using chemotherapy alone is approximately 30%.
Despite a multidisciplinary sequential comprehensive treatment regimen involving
chemotherapy, radiotherapy, surgery, immunotherapy, and hematopoietic stem cell
transplantation (HSCT), the 5‐year survival rate for patients with HR‐NB remains
below 50%[3-5]. Consequently, chemotherapy remains the main method for treating
HR‐NB, and cost‐effective and efficient treatment alternatives for children with
HR‐NB is urgently needed.
The low survival rate in stage 4/M NB can be attributed to multiple factors, including
the difficulty in completely eradicating minimal residual disease (MRD) during
treatment
[6-7] and the emergence of multidrug resistance (MDR) in NB cells
post-chemotherapy
[8-10], which leads to a significant proportion of relapsed or
refractory
NB
cases. Acquired
MDR
is
currently
a
major
barrier
to
the
chemotherapeutic eradication of NB cells and a principal reason for the stagnation in
improving cure rates. In recent years, several innovative research approaches have
been undertaken to address these issues, such as autologous stem cell transplantation
(ASCT) following high-dose chemotherapy and combined anti-GD2 antibody targeted
therapy, which have improved the 5-year event-free survival (EFS) rate for high-risk
NB to approximately 56% [11]. However, the prerequisite medication for NB ASCT,
mafosfamide, is not available in China, and most facilities lack the capabilities to
perform pre-transplant autologous bone marrow purification. The immunotherapy
drug anti-GD2 antibody (Unituxin, dinutuximab), approved by the FDA on March 10,

2015, is extremely costly (at $10,000 per vial, totaling about $200,000 for all five
treatment cycles), and is not readily accessible in China in the short term. The
prohibitive cost and regulatory constraints of Chinese drug policies significantly limit
the implementation of ASCT and anti-GD2 antibody immunotherapy as consolidation
treatments
for
stage
4/M
NB
in
children
within
the
country.
Therefore,
chemotherapeutic treatment remains the main approach for managing high-risk NB in
China at present, and finding effective treatment strategies suitable for the Chinese
context is an ongoing challenge for pediatric oncology specialists.
NB is a highly heterogeneous tumor disease with significant variations in biological
behavior and clinical outcomes. HR-NB patients may experience early widespread
distant metastasis leading to death. However, some patients, such as infants with
localized congenital and stage 4s NB, may see tumors spontaneously shrink or even
disappear, or transform into benign ganglioneuromas, with minimal or no systemic
treatment [12]. This phenomenon of natural regression in NB has captivated many
oncologists and biologists. Although the precise nature of this spontaneous regression
remains unclear, extensive research suggests that the maturation and differentiation of
NB cells may be one of the mechanisms behind this natural regression
[13].
Consequently, finding effective treatments that promote the maturation and natural
regression of highly malignant NB cells is a current focus under therapeutic
challenges. Among the strategies to induce tumor differentiation, retinoids (Retinoic
acid, RA) have been proven to induce differentiation of malignant immature NB cells
into mature ganglion cells or promote apoptosis of tumor cells. However, after
comprehensive treatment using RA to induce differentiation, 40%-50% of high-risk
NB patients still relapse, with RA-resistant minimal residual tumor cells being a
significant factor in recurrence
[14]. Currently, due to the limited availability of
suitable drugs for treating RA-resistant HR-NB in children, finding and testing the
combined use of effective drugs, especially those approved by the China National
Medical Products Administration, is crucial in formulating new treatment plans and
may play a key role in finding new therapeutic strategies for RA-resistant HR-NB.
Arsenic trioxide (ATO) is the major ingredient of a longstanding traditional Chinese

herbal preparation for thousands of years. Clinical studies have demonstrated that low
doses of ATO can induce complete remission in patients with relapsed acute
promyelocytic leukemia (APL) with favorable safety profile, and are now widely
adopted in the clinical management of APL [15]. The utilization of ATO for the
treatment of APL was officially sanctioned by the US Food and Drug Administration
(FDA) in 2000. Furthermore, numerous investigations and clinical trials have
provided substantial evidence supporting the effectiveness of conventional doses of
ATO in eliminating various types of tumor cells, including NB, multiple myeloma,
breast cancer, ovarian cancer, hepatocellular carcinoma, and osteosarcoma, etc[16].
Numerous Phase I and II clinical trials are currently underway to determine the
efficacy and safety of ATO in the clinical treatment of tumors [17-22]. The Memorial
Sloan-Kettering Cancer Center in the United States has preliminarily completed a
Phase II clinical trial on ATO for the treatment of refractory/recurrent solid tumors in
children,
including
NB
(detailed
information
can
be
found
at
https://clinicaltrials.gov/ct2/show/study/NCT00024258?term=neuroblastoma+arsenic
+trioxide&rank=1). Although specific methods and efficacy details have not been
published, these studies suggest that ATO
holds potential for treating MDR NB. The
mechanisms of ATO’s cytotoxic effects on tumor cells are complex, involving the
induction of tumor cell differentiation, inhibition of growth, and promotion of
apoptosis[23]. In APL cells, ATO primarily targets the PML/RARA oncogene for its
anti-tumor effects; in other tumor cells, it acts mainly through Caspase-dependent
apoptosis pathways and down-regulation of the Bcl-2 gene expression. ATO induces
apoptosis
independently
of
P53
gene
expression;
it
works
primarily
by
down-regulating Bcl-2, activating Caspases, generating reactive oxygen species,
disrupting mitochondrial membrane potential, and blocking the cell cycle at G0 or
G2/M phase, thereby promoting tumor cell apoptosis [16]. ATO acts on multiple targets
in NB cells to promote their apoptosis and reduce the likelihood of developing drug
resistance. Amplification of the MYCN gene is a prognostically adverse factor closely
associated with neuroblastoma. The MYCN gene primarily promotes the proliferation
of NB cells through mechanisms such as the transcriptional activation of target genes

like ODC, MCM7, MDM2, MDR1, and PAX3, and by promoting the transition of NB
cells from G1 to S phase. Kim DW et al. [24] found that the anti-tumor effects of
STI-571 (Imatinib Mesylate) and As2O3 are not diminished in NB with MYCN gene
amplification. Yin Hua et al. [25] found that different concentrations of ATO could
reduce the expression of MYCN mRNA in LA-N-5 NB cells, with the most
significant reduction at a concentration of 3.0 μmol/L. The numerous mechanisms of
ATO in treating NB avoid inducing MDR, such as its cytotoxic effects on NB cells
being independent of the P53 gene, and its undiminished anti-tumor effects in
MYCN-positive NB cells, as well as its ability to down-regulate MYCN gene
expression. The most significant advantage of ATO in treating NB is its efficacy in
treating MDR NB and its synergistic effects with various anti-tumor drugs; unlike
traditional chemotherapy, the cytotoxicity of ATO against resistant NB cells does not
significantly decrease under both normoxic and hypoxic conditions, making it a
potential effective drug for future treatment of MDR NB. Currently, there are no
reported clinical studies on the combined use of ATO with chemotherapy in treating
HR-NB internationally or domestically.
The findings
from
our prior
investigations
[26-28] have
established
that
the
administration of ATO exhibits a notable capacity to augment the cytotoxic effects on
NB cells. ATO significantly blocked SK-N-SH cells in G2/M phase; the expression of
drug-resistant proteins (e.g. P-gp) in SK-N-SH cells did not increase with the increase
of ATO concentration and time; and the expression of TrkA and Trk C, which have the
effect of promoting NB differentiation and apoptosis, increased significantly with the
increase of ATO concentration. Our in vitro experiments showed that the IC50 of ATO
in SK-N-SH cell line was 3 umol/L, which is very close to its effective serum
concentration in the clinical treatment of APL, suggesting that the use of ATO in
combination with chemotherapy, which is similar to that used in the treatment of APL,
can be effectively used in the treatment of NB. In addition, further studies showed that
the combination of ATO and chemotherapeutic agents (vincristine, docetaxel,
etoposide or cisplatin) enhanced their cytotoxic effect on SK-N-SH cells; and when
As2O3 was used in combination with M-phase specific chemotherapy (vincristine,

docetaxel), the cytotoxic effect on SK-N-SH cells was enhanced, and when ATO was
used in combination with M-phase specific chemotherapy (vincristine, docetaxel), it
was not effective in treating APL. When ATO was combined with M-phase-specific
chemotherapeutic agents (vincristine, docetaxel), the cytotoxic effect on SK-N-SH
cells was significantly enhanced if ATO was administered first to block the SK-N-SH
cell cycle at the G2/M phase and then M-phase-specific chemotherapeutic agents
were added. When ATO was combined with non-M phase-specific chemotherapeutic
drugs (etoposide and cisplatin), the administration of ATO first did not further
increase the cytotoxic effect on NB cells, but it was still significantly better than that
of the single drug administration.
In summary, to further improve the chemotherapy response rate, event-free survival,
and long-term survival rates for children with HR-NB, there is an urgent need to
identify new therapeutic drugs. Inducing maturation, differentiation, and apoptosis in
NB cells is a key strategy for treating relapsed or refractory NB. ATO has a broad
spectrum of antitumor activities, and its efficacy and safety in treating malignant
tumors have been clinically validated. In vitro studies suggest that ATO can act on NB
cells
through
multiple
mechanisms,
and
its
combined
use
with
traditional
chemotherapy drugs can enhance chemotherapeutic effects. This study primarily
investigates the clinical efficacy and safety of ATO combined with chemotherapy in
treating stage 4/M NB, offering a new treatment approach and option for relapsed or
refractory NB.
2. STUDY OBJECTIVES
2.1 PRIMARY OBJECTIVE
By observing the objective response rate (ORR) of 4/M stage NB treated with ATO
combined chemotherapy at the fourth week after the completion of induction
chemotherapy, and using the current ORR of 4/M stage NB as an external control, this
study aims to evaluate the clinical efficacy of ATO combined chemotherapy in
treating 4/M stage NB.

2.2 SECONDARY OBJECTIVES
2.2.1 To investigate the overall survival rate (OS) of 4/M stage NB treated with ATO
combined chemotherapy.
2.2.2 To investigate the progression-free survival (PFS) of 4/M stage NB treated with
ATO combined chemotherapy.
2.2.3 To investigate the disease control rate (DCR) of 4/M stage NB treated with ATO
combined chemotherapy.
2.2.4 To evaluate the safety of ATO combined chemotherapy in treating 4/M stage
NB.
3. STUDY DESIGN
3.1. DESCRIPTION OF STUDY
This study is a prospective, single-arm, open-lable, multi-center clinical trial. This
study employs the Simon two-stage optimal design, planning to enroll 65 patients (19
patients in the first stage and 40 patients in the second stage) who are ≤14 years old,
have evaluable lesions, and are newly diagnosed with NB classified as stage 4
according to INSS and/or stage M according to INRG. The enrollment is expected to
be completed within 36 months, with follow-up for OS continuing for 36 months after
the last patient is enrolled. The entire study is projected to last 72 months.
The enrolled patients will receive ATO combined with the traditional induction
chemotherapy regimen for stage 4/M NB. The study will calculate the objective
response
rate and
overall
survival
rate
following ATO combined
induction
chemotherapy, and analyze chemotherapy-related adverse reactions. The overall
treatment level of 4/M stage NB reported in the literature will serve as an external
control to evaluate the efficacy and safety of ATO combined chemotherapy for 4/M
stage NB.
Patients will continue the specified treatment until objective disease progression,
symptom deterioration, unacceptable toxicity, death, or withdrawal of consent

(whichever occurs first). Chemotherapy-related adverse reactions will be closely
monitored
and
recorded
throughout
the
treatment.
Comprehensive
disease
assessments will be conducted every 2-3 cycles, at the fourth week after the end of
induction chemotherapy, and at the fourth week after the completion of all treatments.
Following the completion of all treatments, patients will be followed up every 3-6
months to monitor disease and health status until death or the end of the study.
3.2. SELECTION OF STUDY POPULATION
3.2.1 Inclusion Criteria
(1) Untreated Stage 4/M neuroblastoma patients according to the INSS or the INRG
staging system.
(2) Patients not more than 14 years old.
(3) There are measurable lesions.
(4) Guardians agreed and signed informed consent.
3.2.2 Exclusion Criteria
(1) Patients who had suffered from other tumors and received chemotherapy or
abdominal radiotherapy.
(2) Patients with one or more critical organs failure such as heart, brain, kidney failure
and so on.
3.2.3 Discontinuation Criteria
(1) Premature termination of treatment
(2) Non-compliance with treatment regimen, not treating according to staging and
tissue type
(3) Disease progression after completing four cycles of induction chemotherapy
(4) Occurrence of severe adverse events, complications, or special physiological
changes that make continued treatment inadvisable
3.2.4 Case Numbers

Based on the large-sample case analysis reports from multiple medical centers
domestically and internationally, the ORR (including complete and partial responses)
after standard induction chemotherapy for stage 4/M NB is approximately 70%. We
hypothesize that the induction remission rate of ATO combined with chemotherapy
reaching 85% would be considered clinically valuable, using a power of 80% and a
two-sided significance level of 0.05.
According to the Simon two-stage optimal design for single-group clinical trials,
calculated using the Power and Sample Size Program, this study expects to enroll 19
patients ≤14 years old with stage 4/M NB in the first phase. If no more than 14
patients benefit, the trial will be terminated. Otherwise, the second phase will
continue to enroll 40 patients. If the total number of effective cases in both phases
exceeds 46, it will be considered that ATO combined with chemotherapy can benefit
patients. A total of 59 cases need to be enrolled in both phases. Considering the 10%
dropout rate, 65 cases are planned to be enrolled in this study.
3.3. Clinical Pathological Diagnosis and Staging
All cases must have a confirmed pathological histological classification and staging
before starting treatment.
3.3.1 Histological Classification
(1) Neuroblastoma: Undifferentiated; Poorly Differentiated; Differentiated
(2) Ganglioneuroblastoma: intermixed; Nodular
(3) Ganglioneuroma: Maturing subtype; Mature subtype
3.3.2 International Neuroblastoma Staging System (INSS)
Stage
Description
1
Localized tumor with complete gross excision, with / without microscopic residual
disease
Ipsilateral lymph nodes negative for tumor microscopically
Lymph nodes attached to and removed with primary tumor may be positive
2A
Localized tumor with incomplete gross excision
Ipsilateral lymph nodes negative for tumor microscopically

2B
Localized tumor with / without complete gross excision
Ipsilateral, nonadherent lymph nodes positive for tumor
Enlarged contralateral lymph nodes must be negative for tumor microscopically
3
Unresectable unilateral tumor infiltrating across the midline (midline is defined as the
vertebral column)
with / without regional lymph node involvement
OR
Localized unilateral tumor
With contralateral regional lymph node involvement
OR
Midline tumor
With bilateral extension by infiltration (unresectable) or by lymph node involvement
4
Disseminated tumor to distant lymph nodes, bone, bone marrow, liver, skin and/or other
organs (except as defined for stage 4S)
4S
oLocalized primary tumor (as defined for stage 1, 2A, or 2B) with dissemination limited
to skin, liver and/or bone marrow (<10% of nucleated cells)
Only in infants <1 year in age
3.4. Pre-treatment Examination
3.4.1 Medical History Collection
In addition to the current medical history, it is necessary to include past health status,
family history of cancerous diseases, and social and environmental exposure to
harmful physical and chemical factors. Check for any congenital malformations.
3.4.2 Physical Examination
Height, weight, body surface area. Blood pressure, examination of the head, face,
limbs,
chest,
cardio-pulmonary,
superficial
lymph
nodes,
abdominal
masses,
reproductive organs, etc.
3.4.3 Laboratory Tests
(1) Routine tests: complete blood count, blood type, routine urine analysis (including
occult blood), stool routine.
(2) Biochemical tests: liver and kidney functions, electrolytes, coagulation function,
blood NSE, and 24-hour urine VMA (for young children who have difficulty retaining
urine, a single 20ml urine VMA/Cr ratio may be performed).
(3) Cardiac function tests: ECG, UCG, and cardiac enzyme tests.

(4) Imaging studies of the lesion include: X-ray, CT or MRI scans of the lesion site,
head, chest, and abdomen (plain and enhanced), whole-body bone scan, and if
necessary, PET-CT to determine the extent and location of tumor invasion.
(5) Routine human PPD skin test, bone marrow examination (single or double iliac
crest).
(6) Lesion biopsy and pathological type report; if surgical biopsy is not possible at
initial diagnosis, one of the following two diagnostic criteria is needed: a. Presence of
metastatic NB cells in bone marrow with corresponding immunohistochemistry and
morphological features; b. Imaging supports NB diagnosis along with significantly
elevated 24-hour urine VMA (or single urine VMA/Cr ratio).
(7) N-myc gene testing: send bone marrow sample affected by the tumor or excised
tumor pathological sample.
(8) Arsenic baseline value: Collect any urine sample before arsenic treatment and send
the same urine sample for arsenic testing (conducted by KingMed Diagnostics) and
urine creatinine (tested by Sun Yat-sen University Sun Yat-sen Memorial Hospital),
reported as: μg arsenic/g creatinine
3.5. Treatment Protocol
The induction treatment for stage 4/M neuroblastoma (NB) includes chemotherapy,
surgery, and radiation therapy. The induction protocol consists of 9 chemotherapy
cycles: Cycles 1, 2, 4, and 6 follow the CAV regimen (Cyclophosphamide +
Pirarubicin + Vincristine); Cycles 3, 5, and 7 follow the PVP regimen (Cisplatin +
Etoposide); Cycles 8 and 9 follow the CT regimen (Cyclophosphamide + Topotecan).
The chemotherapy intervals for the CAV and PVP regimens are 2-3 weeks; for the CT
regimen, the interval is 3-4 weeks. Surgical operations are generally scheduled
between cycles 4 and 5 (as shown in Table 1). Radiation therapy, for children aged ≥1
year, is generally administered within one month after surgery, and chemotherapy is
continued at the corresponding intervals after the completion of radiation therapy; for
children who do not require radiation therapy (aged < 1 year), chemotherapy is
administered according to the diagram. Some NB patients, after completing 4 cycles

of chemotherapy (3 CAV regimens + 1 PVP regimen) and undergoing a PET-CT scan
that shows no solid tumor lesions, do not require surgery and will continue the
remaining chemotherapy cycles at the corresponding intervals.
Table 1. Protocol of the Induction Chemotherapy
Table 2.Composition of Chemotherapy Regimen
ATO
ATO
0.16 mg/kg/day, iv drip, PI > 4h
d1 to d10
CAV Regimen
Cyclophosphamide (CTX)
1.2 g/m²/day, iv drip, PI = 3h
d3-d4
Pirarubicin (THP)
25 mg/m²/day, iv drip, PI > 3h
d3-d5
0.022 mg/kg/day or 0.67 mg/m²/day,
iv drip, PI = 2h
d3-d5
Use the lower of the two calculation
methods; maximum daily dose ≤0.67
mg, total dose over 3 days > 2 mg
d3-d5
Vincristine (VCR)
0.022 mg/kg/day or 0.67 mg/m²/day,
iv drip, PI = 2h
d3-d5
PVP Regimen
Cisplatin (DDP)
50
mg/m²/day,
iv
drip,
PI
=
6h
(light-protected)
d3-d6
Etoposide (VP-16)
200 mg/m²/day, iv drip, PI > 4h
d3-d5
CT Regimen
Cyclophosphamide (CTX)
1.2 g/m²/day, iv drip, PI = 3h
d3-d4
Topotecan
2
mg/m²/day,
iv
drip,
PI
=
24h
(light-protected)
d3-d5
Precautions:
(1) Entry criteria for starting chemotherapy: Neutrophils > 0.5 × 10^9/L, Platelets >
50 × 10^9/L.
(2) Entry requirements for treatment with ATO: a. Serum potassium: 3.5-5.3 mmol/L,
serum magnesium: 0.74-1.03 mmol/L, serum calcium: 2.02-2.60 mmol/L; b. Total
serum bilirubin ≤1.5 mg/dL, serum creatinine ≤1.5 mg/dL; c. 12-lead ECG showing:
QT interval < 0.5 s.
(3) Each chemotherapy cycle starts with two days of ATO chemotherapy alone,
followed by combination with the standard induction chemotherapy regimens (CAV,

PVP, CT) starting from the third day. ATO is used for a total of 10 days per cycle
(d1–d10).
(4) Dosage and administration of ATO: 0.16 mg/kg.d, mixed with 5% glucose solution
or 0.9% normal saline 250-500ml, intravenous drip over more than 4 hours;
concurrently administer vitamin C to reduce adverse reactions and enhance its
effectiveness in the body: vitamin C 0.5-1.0g, mixed in 5% glucose solution 100-250
ml, administered simultaneously with ATO by intravenous drip.
(5) In the CAV regimen, Cyclophosphamide (CTX) requires pre-hydration and
alkalinization half a day in advance, along with Mesna (administered concurrently
with CTX, daily dose of 1200 mg/m^2, divided into three doses, continued for 2 days,
total dose equal to 100% of the CTX dose), alkalinization, hydration, diuresis,
antiemetic etc., starting from the first day of CTX, continuous hydration and
alkalinization for at least 5 days; Pirarubicin (THP) dissolved in 5% glucose solution
250-500 ml IV drip over more than 3 hours, with antiemetics; Vincristine (VCR)
dissolved in normal saline 100ml IV drip over 2 hours, maximum daily dose ≤0.67
mg, total dose over 3 days ≤2mg.
(6) In the PVP regimen, starting from the day of Cisplatin (DDP) administration,
continue hydration and alkalinization for at least 7 days, fluid replacement 3000
ml/m2.d, 20% mannitol for diuresis, monitor urine volume, check routine urine;
before administering DDP, give mannitol (20% mannitol 1.25-5.0 ml/kg + normal
saline 100-250 ml, rapid IV drip), start hydration treatment 2 hours before, when urine
output reaches 250 ml/h, start DDP administration (in normal saline, concentration not
exceeding 1mg/ml, IV drip = 6h, protect IV bottle from light); if cardiovascular
function is compromised (output < 45%), appropriately reduce hydration volume and
limit the single dose of DDP; antiemetics; monitor for nephrotoxicity and severe,
persistent electrolyte disturbances caused by DDP, and manage accordingly if
significant hyponatremia, hypomagnesemia, hypocalcemia, or hypokalemia occur,
which can happen days after DDP treatment; for allergic reactions caused by DDP,
treatment with epinephrine, antihistamines, and corticosteroids is effective; Etoposide
(VP16) dissolved in normal saline 250-500 ml (ensure drug preparation concentration

limits, not exceeding 0.25mg/ml) IV drip over more than 4 hours, used for 3
consecutive days, avoid using glucose solution for preparation, slow drip required.
(7) In the CT regimen, Cyclophosphamide (CTX) requires pre-hydration and
alkalinization half a day in advance, along with Mesna (administered concurrently
with CTX, daily dose of 1200 mg/m^2, divided into three doses, continued for 2 days,
total dose equal to 100% of the CTX dose), alkalinization, hydration, diuresis,
antiemetic etc., starting from the first day of CTX, continuous hydration and
alkalinization for at least 5 days; Topotecan dissolved in normal saline 250ml–500ml
IV drip over 24 hours (continuous IV drip, best protected from light), used for 3
consecutive days, with antiemetics.
(8) Audiometric testing (ENT examination items: a. Pure tone audiometry b. Acoustic
impedance + stapedius muscle reflex attenuation c. Acoustic reflex)
3.6. Adverse Reactions and Management
(1) Leukocytosis syndrome: A few patients may experience an increase in white
blood cells, appearing 2-3 weeks after medication initiation. There is no need to stop
the treatment; white cell counts can decrease on their own or can be reduced with oral
hydroxyurea within a week.
(2) Digestive system: Nausea, vomiting, anorexia, abdominal pain, and diarrhea are
common adverse reactions. Symptomatic treatment is effective, and these symptoms
usually resolve after stopping the medication. Some patients may experience liver
damage, including elevated transaminases and jaundice; liver-protective medication
can be used, and liver function typically returns to normal after stopping the
medication.
(3) Fluid retention: Patients may experience weight gain, pleural effusion, pericardial
effusion, and facial edema during treatment.
(4) Urinary system: Acute renal failure is rare, but changes in kidney function may
occur and generally recover after stopping the medication.
(5) Nervous system damage: Symptoms such as polyneuritis and multiple radiculitis
may occur about 10-20 days after medication use. Patients may experience limb pain,

numbness, and a progression from hypersensitivity or abnormal sensations to delayed
or absent pain, temperature, and touch sensitivity, even leading to sensory ataxia.
Additionally, there may be limb weakness, distal muscle atrophy, and significant
autonomic disturbances. Arsenic-induced peripheral neuropathy is indistinguishable
from general ataxia. About 34% of patients experience varying degrees of transient
cerebrovascular spasm-induced headaches early in the medication regimen.
(6) Cardiovascular system: Symptoms may include palpitations, chest discomfort,
and ECG changes, including sinus tachycardia, ST segment depression, T-wave
inversion or flattening, prolonged PR interval, or complete atrioventricular block,
though these are generally reversible; prolonged QT interval and ventricular
arrhythmias based on this may occur.
(7) Skin dryness, erythema, or pigmentation.
(8) Differentiation syndrome: Common in the early stages of arsenic-induced
treatment,
characterized
by
elevated
white
blood
cells,
fever,
weight
gain,
musculoskeletal pain, respiratory distress, pulmonary interstitial infiltration, pleural
effusion, pericardial effusion, skin edema, hypotension, acute renal failure, and even
death; severe leukocytosis can also lead to embolisms in the lungs, brain, and other
organs. Prevention and management: Monitor blood counts daily during the early
induction phase. Whether at the start of induction or during, if WBC > 15 × 10^9/L,
administer hydroxyurea (HU) 100 mg/kg.d (based on WBC), divided into 2-3 doses,
and use dexamethasone (DXM) 0.3-0.5 mg/Kg.d if symptoms occur, until they
disappear. For WBC > 30 × 10^9/L, in addition to HU, it is recommended to use
DXM to prevent differentiation syndrome.
Other adverse reactions can be treated symptomatically, and most symptoms
disappear after stopping the medication. Some children may experience significant
gastrointestinal reactions at the start of each treatment cycle, possibly accompanied by
mild headaches, which typically lessen or disappear after 2-3 days. It is recommended
to halve the dose for the first 2-3 days, then adjust to the full dose or reduce to 3/4
dose depending on tolerance. To minimize gastrointestinal reactions, doses should be

taken orally at least three times a day, and antiemetic medication such as
metoclopramide may be taken half an hour beforehand to alleviate symptoms of
nausea and vomiting. For individual cases with severe gastrointestinal reactions that
remain intolerable after at least 3 days and additional antiemetics, discontinuation
from the study group is recommended.
3.7. Observation Indicators and Follow-up
3.7.1 Observation Indicators During Chemotherapy
Patients need to meet the enrollment criteria. Before each chemotherapy cycle,
complete blood count, routine biochemistry, and ECG/echocardiogram are required.
During chemotherapy, monitor and record in detail the side effects such as bone
marrow
suppression,
gastrointestinal
reactions,
liver
and
kidney
damage,
cardio-pulmonary toxicity, neurotoxicity, allergic reactions, and local toxicity. After
each chemotherapy cycle, perform 24-hour urine VMA and blood NSE tests. Every
2-3 cycles, perform imaging studies of the lesion (abdominal CT and/or chest CT),
and assess the tumor status through bone marrow or peripheral blood minimal residual
disease.
3.7.2 Follow-up After Treatment Completion
(1) Four weeks after the completion of all ATO combined chemotherapy regimens,
conduct a comprehensive re-examination including complete blood count, routine
biochemistry, ECG, echocardiogram, chest and abdominal CT, or whole-body PET-CT,
and human arsenic content tests (hair, nails, 24-hour urine).
(2) Within the first year after all treatments are completed, perform abdominal
ultrasound, chest X-ray, and ECG every three months, and conduct growth and
development physical examinations (measuring height and weight); every six months,
re-examine abdominal CT and echocardiogram, complete blood count, and routine
biochemistry (liver and kidney functions and electrolytes), and conduct growth and
development physical examinations.
(3) Within the second year after all treatments are completed, every six months,

re-examine abdominal ultrasound or CT, chest X-ray, echocardiogram, complete
blood count, and routine biochemistry (liver and kidney functions and electrolytes),
and conduct growth and development physical examinations (measuring height and
weight).
(4) From the third year after all treatments are completed, perform annual
re-examinations
including
abdominal
ultrasound
or
CT,
chest
X-ray,
ECG,
echocardiogram, complete blood count, and routine biochemistry (liver and kidney
functions
and
electrolytes),
and
conduct
growth
and
development
physical
examinations (measuring height and weight).
3.8. Criteria for Efficacy Evaluation
WHO criteria for efficacy evaluation are adopted.
3.8.1 Measurable Lesions
(1) Complete Response (CR): All lesions completely disappear, maintained for at least
4 weeks.
(2) Partial Response (PR): For bi-dimensional measurable lesions, the sum of the
products of the maximum perpendicular diameters of all lesions (taking the largest
diameter and the perpendicular diameter, multiplying them to get the maximum
perpendicular product, and then summing these products for all lesions) reduces by
more than 50%, maintained for at least 4 weeks; for uni-dimensional measurable
lesions, the sum of the maximum diameters of all lesions reduces by more than 50%,
maintained for at least 4 weeks.
(3) No Change (NC): For bi-dimensional measurable lesions, the sum of the products
of the maximum perpendicular diameters reduces by less than 50% or increases by no
more than 25%, maintained for at least 4 weeks; for uni-dimensional measurable
lesions, the sum of the maximum diameters reduces by less than 50% or increases by
no more than 25%, maintained for at least 4 weeks. NC can only be evaluated after at
least two treatment cycles (6 weeks).
(4) Progressive Disease (PD): One or more lesions increase by more than 25%, or

new lesions appear. If new pleural or peritoneal effusions are found with cancer cells
in cytology, it should be judged as PD.
3.8.2 Evaluative but Non-Measurable Lesions
(1) CR: All lesions completely disappear, maintained for at least 4 weeks.
(2) PR: Estimated tumor size reduction by more than 50%, maintained for at least 4
weeks.
(3) NC: No significant change in lesions after at least two treatment cycles (6 weeks),
estimated tumor reduction by less than 50% or increase by no more than 25%.
(4) PD: New lesions appear, or estimated tumor size increases by more than 25%.
3.8.3 Bone Metastasis Lesions
(1) CR: Osteolytic lesions disappear, bone scans return to normal, maintained for at
least 4 weeks.
(2) PR: Partial reduction or calcification of osteolytic lesions, or reduced density of
osteoblastic lesions, maintained for at least 4 weeks.
(3) NC: No significant change in lesions, can only be evaluated as NC after at least 8
weeks of treatment.
(4) PD: New lesions appear, or existing bone lesions significantly enlarge. However,
bone compression, pathological fractures, or bone healing should not be the sole
criteria for efficacy evaluation.
3.8.4 Non-Evaluable Lesions:
(1) CR: All lesions completely disappear, maintained for at least 4 weeks.
(2) NC: No significant change in lesions, estimated tumor reduction by less than 50%
or increase by no more than 25%, maintained for at least 4 weeks.
(3) PD: New lesions appear, or estimated tumor size increases by more than 25%. If
effusions in cavities increase without other lesion progressions, it should not be
evaluated as PD

3.9. Safety Evaluation Criteria
The intensity of adverse events will be graded according to the grading methods
recommended by the NCI CTC (version 3.0, 2003-12-12) Common Toxicity Criteria
of the National Cancer Institute.
3.10. Statistical Analysis Methods
This study is designed as a single-group clinical trial, with results primarily described
using statistical descriptions, and no hypothesis testing will be conducted. The
efficacy indicators will be calculated with α = 0.05 (two-sided) to compute the 95%
CI.
(1) Efficacy Indicators: The remission rate at the fourth week after the end of
induction chemotherapy will be the trial evaluation indicator. Additionally, it will
serve for further research on the efficacy and safety of arsenic trioxide combined with
chemotherapy. The overall survival rate (OS), event-free survival rate (EFS), and
progression-free survival time (PFS) will also be calculated and the corresponding
survival curves will be plotted.
(2) Safety
Data:
Detailed
descriptions
of
cases
with
hematological
or
non-hematological toxicity reactions will be provided. The incidence rates of different
events will be calculated, and the composition ratio of the severity of each event will
be computed.
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