Background Bladder cancer is a malignant tumor originating from the uroepithelium of
the bladder, accounting for the first place in the incidence of genitourinary tumors
in China. Among them, bladder uroepithelial cancer is the most common, accounting
for more than 90% of bladder cancers. According to the depth of tumor invasion into
the bladder and the prognostic characteristics, bladder cancer is clinically
classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive
bladder cancer (MIBC), with NMIBC as the main type, accounting for about 75% of all
bladder cancer patients .
NMIBC is usually treated with Transurethral Resection of Bladder Tumor (TURBt).
Despite being in the early stages of the disease, NMIBC has a high recurrence rate,
with studies reporting a 5-year recurrence rate of approximately 70% . Therefore,
postoperative bladder perfusion chemotherapy has become a key therapeutic measure to
prevent recurrence of bladder cancer.For more than 60 years, people have been
exploring more effective bladder perfusion drugs and methods.In 1961, Thiotepa was
used for bladder perfusion therapy, and gained a certain degree of efficacy in
preventing the recurrence of superficial tumors of the bladder.In 1976, Morales
believed that bladder cancer was related to immune deficiencies, and accordingly BCG
bladder perfusion therapy has been extremely successful, creating a new way of
immunotherapy for bladder tumors, which has been widely valued and is still
considered the most effective bladder perfusion drug. However, BCG instillation can
cause serious side effects, including urethral and prostatic granulomas, bladder
irritation, fever, hematuria and other local manifestations and systemic
influenza-like symptoms, resulting in intolerance and interruption of treatment in
some patients, which has limited the wide clinical application of BCG. Studies have
shown that intravesical instillation of chemotherapeutic agents in the bladder can
reduce the near-term recurrence rate of superficial bladder cancer by about 15%-20%
and the long-term recurrence rate by about 6% . Currently there are many choices of
chemotherapeutic agents in bladder instillation, including (1) pirenzolubicin; (2)
gemcitabine; (3) mitomycin; (4) epirubicin; (5) doxorubicin; (6) alternating
bladder-infusion chemotherapy with gemcitabine and pirenzolubicin; and (7) other
combined-infusion chemotherapies. However, due to the wide variety of
chemotherapeutic drugs, drug selection mostly relies on clinical experience or
certain research results, there is no uniform standard, and it is blind, empirical,
and randomized. Chinese urological disease diagnosis and treatment guidelines also
did not provide the selection of perfusion drugs, and individual patients for some
chemotherapeutic drugs show natural resistance, when the clinic determines that the
patient is not sensitive to the application of the drug, the drug has produced
serious toxic side effects, and even lead to the phenomenon of multi-drug resistance
(multi-drug resistance, MDR) produced, so that the clinical The recurrence rate is
still as high as 36%-44%, and at the same time, the tumor of this group of patients
progresses rapidly, losing the opportunity to re-select the treatment method.
Therefore, how to avoid the selection of primary drug-resistant drugs and directly
choose drugs with high sensitivity to achieve individualized chemotherapy has become
a hot spot in the research of bladder perfusion chemotherapy.
The realization of precision tumor therapy greatly depends on the detection of drug
sensitivity. Through precise individualized chemotherapeutic drug screening
experiments, the most effective and least toxic therapeutic regimen can be judged
for each patient before the start of treatment, so as to propose a chemotherapeutic
regimen for a single patient is a new direction of research to realize the precision
treatment of bladder cancer and to improve the efficiency of bladder cancer
chemotherapy. Previously, the more commonly used preclinical models are traditional
tumor cell lines and human-derived tumor tissue xenografts (PDX). Tumor cell line
culture method is simple but insufficient to simulate the growth state of tumor
cells in patients, and the drugs screened by its drug screening system have low
value for clinical application.PDX, although it can simulate in vivo tumor
characteristics and preserve the tumor microenvironment, has obvious limitations
such as low stable tumorigenicity, long modeling and evaluation period (half a year
to one year), time-consuming and laborious, and it is difficult to generate and use
for high-throughput drug screening. Therefore, the development of drug-sensitivity
assay models that can mimic the heterogeneity and complexity of bladder cancer has
become necessary in order to develop more personalized therapeutic and preventive
strategies to minimize risk and optimize the effectiveness of medical interventions
by targeting the unique genetic, environmental and lifestyle characteristics of
individuals.
Patient-Derived Organoids (PDO) are 3D organoid structures formed by stem cells
self-assembled in vitro, which can be differentiated into multiple organ-specific
cell types and can exhibit cell-cell and cell-surrounding matrix interactions and
spatial location patterns, recreating in vitro some of the key functions and
structures of real organs, and having a stable phenotype. structures with stable
phenotypic and genetic characteristics. Compared with two-dimensional tumor cell
lines and PDX, tumor organoids can be cultured directly using the patient's own
tissues, and at the same time, these organoids can well replicate some of the key
characteristics of the primary tumors, retain the pathomorphology and biological
mechanisms of the patient's tissues, and preserve the heterogeneity of the tumor
tissues and a more realistic tumor microenvironment, as well as having a short
growth cycle. It is helpful for its use in clinical cancer patients for drug
sensitivity testing of radiotherapy drugs, molecular targeting drugs, anti-tumor
antibodies and other drugs, to predict the patient's responsiveness to drugs, with
the potential to assist in clinical treatment decisions.
In 2018, Science reported a study on the use of metastatic gastrointestinal
tumor-like organs for drug sensitivity testing, which comparatively analyzed the
differences in sensitivity between 21 clinical patients and their corresponding PDOs
to a series of targeted and chemotherapeutic drugs, and the results showed strong
consistency between the two. In comparison with the actual patient outcomes, the
PDOs were well predicted (sensitivity 100%, specificity 93%, positive predictive
value 88%, and negative predictive value 100%).20 In 2020, Yao Y et al constructed
96 rectal cancer organoids using biopsies from 112 cases of locally advanced rectal
cancer, and selected 80 of them to test their response to radiotherapy, and the
results showed that rectal cancer organoids were sensitive to radiotherapy and the
patient's clinical response. The results showed that the sensitivity of rectal
cancer organoids to radiotherapy was highly consistent with the clinical response of
patients (sensitivity 78%, specificity 92%, accuracy 84%). Subsequently, in tumors
such as gastric cancer and breast cancer, the concordance between PDO and tumor
patients' response to drugs was also found. Yan HHN et al constructed a gastric
cancer organoid library using tumor tissues, paracancerous tissues and lymph node
metastases from 34 gastric cancer patients. Two of them developed tumor metastases
and underwent a combination of cisplatin and 5-FU after surgery, both of which
responded well. The other case received chemotherapy before surgery and did not
respond to capecitabine after surgery. Examination of the sensitivity of the
corresponding compounds of PDO in these three cases showed that the drug sensitivity
of PDO was in perfect agreement with the clinical response of each patient.Guillen
KP et al constructed PDX and PDO using tumor samples from endocrine
therapy-resistant, relapsed, and metastatic breast cancer patients, and these
samples were examined histomorphologically, genomically, and drug sensitivity. The
results showed that both breast cancer PDX and PDO were highly reductive of the
histobiological and genomic properties of their source tumors, and both responded
consistently to anti-tumor drugs. A patient with triple-negative breast cancer in
stage IIA in this study developed liver metastases about 1 year after undergoing
preoperative chemotherapy and surgical treatment. The investigators subjected the
constructs PDO and PDX to ex vivo drug sensitivity testing and found that the
microtubule inhibitor eribulin had the best therapeutic effect. Based on this
result, patients were instructed to undergo treatment with eribulin, which resulted
in complete remission of liver metastases for nearly 5 months after dosing. These
studies confirm to some extent the possibility of PDO to guide the medication of
patients with clinical tumors. It has also been shown that by comparing the
difference in response to drugs between normal-like organs and PDO, it has been
found that drugs with high selectivity help to reduce toxic side effects in clinical
patients. Thus, it is clear that drug sensitivity testing by PDO to discover the
most appropriate drug regimen will help to improve the clinical efficacy of tumor
patients, reduce toxic side effects, risk of drug resistance, and chances of tumor
recurrence, and maximize the benefits to patients.
Therefore, relying on patient-derived bladder cancer organoid models, we will carry
out non-muscle invasive bladder cancer perfusion chemotherapy drug sensitivity
testing experiments, establish a standardized drug sensitivity testing system for
bladder cancer organoids, and formulate screening standards for drug sensitivity
testing of bladder cancer organoids, so as to screen the optimal drug combination
regimen, assist in the clinical development of new individualized treatment
protocols, and carry out multi-center clinical validation to achieve a true
"Substitute drug testing".
Research questions and objectives Research questions: This study compares the
one-year tumour-free recurrence survival rate and the three-year tumour-free
recurrence survival rate of patients in the organoid-sensitive drug infusion group,
the organoid-unsensitive drug infusion group, and the BCG infusion group by means of
a cohort study, and the difference in the recurrence rate among the three groups is
compared by one-way K-M survival analysis. Assessment to evaluate the value of
tumour-like organ drug sensitivity assays in guiding individual perfusion
chemotherapy for bladder cancer.
Research objective: to assess the application value of tumour-like organ drug
sensitivity experiments in guiding individual perfusion chemotherapy for bladder
cancer by observing the clinical efficacy of tumour-like organ drug sensitivity
method in guiding postoperative perfusion chemotherapy for bladder cancer patients.
See Outcome Measures for details
Research methodology 3.1 Study design This protocol is based on the Technical
Guidelines for Clinical Trials of Antineoplastic Drugs, and adopts a multicentre,
cohort study.
3.1.1 Grouping See Groups and Interventions for details 3.1.2 Bladder cancer organoid
preparation
Bladder cancer sample collection The sample collection process is in accordance with
hospital ethics and signed informed consent. Patients enrolled in the experiment
must be pathologically diagnosed with bladder cancer, and the size of tumour samples
taken out surgically should be larger than 0.8 cm*3 as far as possible, for
pathological verification, organoid culture, drug sensitivity test, and so on.
Tumour tissues should be collected with attention to tissue ischemia time and
cryopreservation, and the collection time should be within 5 min when the tissues
are removed from the human blood circulation, so as to avoid stress necrosis of the
tumour tissues due to early hypoxia.
Bladder cancer organ preparation In the process of tissue processing, physical
crushing and collagenase digestion were used to process the tissue samples, and the
processed and digested organoids needed to be filtered through cell sieves of
different diameters before culture, so as to make the obtained organoids uniform in
size and facilitate the unification of standards. Then they were planted in
three-dimensional matrix gel and cultured in special medium.
Expansion and culture of bladder cancer organoids The growth status of the organoids
was observed microscopically, and immunohistochemistry and immunofluorescence were
used to characterize the organoids, to confirm the presence of tumour cells and to
evaluate their morphology. Bladder cancer carcinoid organs were passaged and frozen.
3.1.3 Bladder cancer tumour organoid drug sensitivity testing After observing the number
of organoids under microscope to reach the number that can be subjected to drug
sensitivity testing, the organoid medium was aspirated and discarded. Add 3 mL of
digestive solution to the petri dish, blow the droplets apart, digest at 37℃, blow the
organoids and observe under the microscope, when the organoids are digested into a
homogeneous cell mass containing 2-3 cells, add 2 times the volume of the digestive
solution to terminate the digestion. 1500 rpm, centrifuge the cells for 3 minutes, and
then aspirate and discard the supernatant.
Resuspend the cells with medium, try not to produce air bubbles, and dilute the medium to
gel 1:1.5 ratio, then inoculate the mixed gel at 10uL/well with a single-channel pipette
into pre-cooled 384-well plates with 3,000 organoids per well (this operation was
performed on ice). After mixing the medium with the gum at a ratio of 1:1 (40uL:40uL),
the medium was dispensed into the 384-well plate at 10uL/well as a blank group. After
gently tapping the 384-well plate so that the gel droplets lay flat on the bottom of the
wells, the plate was placed in an incubator for 30 min to allow the gel droplets to
solidify and pre-warmed BCOs medium was added at 40 uL/well. To the drug test wells as
well as to the empty wells around the control wells, 40 uL of PBS solution was added and
placed in a carbon dioxide incubator to prevent the liquid from evaporating and causing
the gel drops to dry out.
After 3 days of incubation and observation, microscopic observation of the status of the
sample in each well was carried out for half-volume fluid exchange.
Design the drug sensitivity test protocol according to the requirements of the following
table, set up the control group, blank group and drug test group, the drug was dissolved
according to the instructions and diluted 4-fold into 6 concentrations with organoid
medium, respectively; using luminescent cell viability assay, the drug was tested at
different concentrations of the semi-inhibitory concentration (IC50), inhibition of
tumour cells (IR), the degree of deviation of the IC50 from the blood drug concentration,
AUC ( area under the dose-response curve), the evaluation criteria of the results of the
drug sensitivity assay take IC50 and IR as the main indexes, together with the degree of
deviation of IC50 from the blood drug concentration and the AUC results as the auxiliary
judgement (for details, please refer to 3.10.3), to assess the effects on the survival
and proliferation of the class of organisms, and to give the clinical guidance of the use
of the drug.
Table 1 Methods for grouping drug sensitivity tests Grouping method of drug sensitivity
testing Blank group: No organoid, no drug added, 6 replicate wells per plate. Control
group: Inoculated with organoids, no added drug exposure. Drug test group: Inoculated
with organoid, add drug exposure. 3.1.4 Chemotherapy drug combination regimens Table 2
Perfusion chemotherapy drugs Gemcitabine □ Pirorubicin □ Epirubicin □ Silvestrol □
Adriamycin(doxorubicin) □ 3.2 Study site and study population (source and inclusion
exclusion criteria) See study design for details 3.3 Study Variables (Factors) and
Measurements The positive control group was not subjected to organoid culture as well as
organoid drug sensitivity testing.
The remaining patients were subjected to organoid drug sensitivity testing in accordance
with the drug sensitivity testing protocol, using luminescent cell viability assay to
test the semi-inhibitory concentration (IC50) of the drug, inhibition rate (IR) of the
tumour cells, the degree of deviation of the IC50 from the blood drug concentration, and
the AUC (the area under the dose-response curve) at different concentrations, and the
evaluation criteria of the results of drug sensitivity testing were based on the IC50,IR
as the main indexes with the IC50 deviation from blood drug concentration, AUC results as
auxiliary judgement (see 3.10.3 for details), to assess its effect on the survival and
proliferation of class organs.
Patients who underwent organoid culture and drug sensitivity testing underwent bladder
instillation of chemotherapeutic agents according to the experience of clinicians. The
five perfused drugs included gemcitabine, piroxicam, epirubicin, mitomycin, and
adriamycin (doxorubicin). According to the sensitivity of their perfusion drugs in
organoid drug sensitivity test, they were divided into organoid sensitive drug perfusion
group and organoid non-sensitive drug perfusion group. Perfusion regimen: including
induction perfusion chemotherapy (4-8 weeks after surgery, 1 time per week, 4 times in
total) and maintenance perfusion chemotherapy (1 time per month, 11 times in total).
BCG infusion regimen: 6 induction infusions followed by 3 booster infusions once every 2
weeks to maintain a good immune response, followed by 10 maintenance infusions once a
month for a total of 19 infusions over 1 year.
Clinical measurements: the two groups of patients in the organoid-sensitive drug
perfusion group and the organoid-non-sensitive drug perfusion group were followed up and
analysed for the primary and secondary study endpoints, and one-way Kaplan-Meier survival
analysis was performed to compare the differences in recurrence rates between the two
groups. The COX risk proportion model was used to analyse the recurrence risk ratio of
patients' gender, age, tumour stage and grade between the two groups.
3.4 Study outcomes RECIST1.1 criteria were used for efficacy evaluation, and Choi
criteria and mRECIST criteria were used as auxiliary efficacy evaluation criteria.
Primary study endpoints: one-year tumour recurrence-free survival rate, three-year tumour
recurrence-free survival rate.
Secondary endpoints: one-year tumour progression-free survival rate, three-year tumour
progression-free survival rate.
3.5 Follow-up The two groups of patients in the organ-like sensitive drug perfusion group
and the organ-like non-sensitive drug perfusion group were followed up and analysed for
the primary and secondary study endpoints.
Cystoscopy is the method of choice when reviewing patients with NMIBC and there is no
non-invasive alternative to cystoscopy. Therefore, NMIBC follow-up should be based on
regular cystoscopy. If suspicious lesions of the bladder mucosa are found during the
examination, biopsy should be performed to clarify the pathological findings. Urine
exfoliative cytology, CT/CTU or MRI/MRU are performed if necessary, but none of them can
completely replace cystoscopy.
The cystoscopy review plan: low-risk NMIBC is done once at 3 months and 12 months after
surgery in the 1st year, and once a year thereafter until the 3rd year; intermediate-risk
NMIBC is done once at 3 months, 6 months, and 12 months after surgery, every 6 months in
the 2nd year, and every year thereafter until the 3rd year; high-risk NMIBC is done every
3 months in the first 2 years, and every 6 months thereafter until the 3rd year.
3.6 Sample size A sample size of 293 cases was sufficient to fulfil the requirements for
the study endpoints.
There were 73 cases in the organoid non-sensitive drug perfusion group and 110 cases each
in the organoid sensitive drug perfusion group and positive control group.
Calculation basis:
Calculation of sample size for the one-year tumour-free survival rate study
endpoint: the one-year tumour-free survival rate is expected to be 73% in the
organoid-sensitive drug infusion group, and 50% in the organoid non-sensitive drug
infusion group, with unilateral α=0.05, β=0.2, and the ratio of the sample sizes of
the two groups is 1 (experimental group: control group), the sample sizes of the two
groups are 55 cases in the experimental group, 55 cases in the control group, and
110 cases in the positive control group. 55 cases in the control group, totalling
110 cases, and considering 20% shedding, a total sample size of 138 cases is
required.
Calculate the sample size according to the endpoint of the three-year tumour-free
survival rate study: it is expected that the three-year tumour-free survival rate of
the organoid-sensitive drug infusion group will be 63%, and the three-year
tumour-free survival rate of the organoid-unsensitive drug infusion group will be
40%, and the unilateral α=0.05, β=0.2, and the ratio of the sample sizes of the two
groups will be 1 (experimental group: control group), so that the sample sizes of
the two groups will be calculated as 58 cases in the experimental group, 58 cases in
the control group, and 11 cases in the control group, taking into account 20%
shedding, and a total sample size of 138 cases will be required. 58 cases in the
control group, totalling 116 cases, and considering 20% shedding, a total sample
size of 146 cases was required.
According to the results of the literature, the one-year tumour-free survival rate
of the positive control group is 75%, and according to the pre-test, the one-year
tumour-free survival rate of the organoid-sensitive drug infusion group is expected
to be 73%, and the non-inferiority cut-off value is 0.2 (control group-experimental
group), and the ratio of the sample sizes of the two groups is one with the
unilateral alpha being 0.05 and beta being 0.2, the sample size of the two groups is
calculated as 74 cases of the organoid sensitive drug infusion group, 74 cases of
the positive control group, and a total of 116 cases of the experimental group. 74
cases, and 74 cases in the positive control group, for a total of 148 cases.
Considering 20% shedding, the total sample size required is 186 cases.
According to the results of the literature, the three-year tumour-free survival rate
of the positive control group is 65%, and according to the pre-test, the three-year
tumour-free survival rate of the organoid-sensitive drug infusion group is expected
to be 63%, and the non-inferiority cut-off value is 0.2 (control group-test group),
and the ratio of the sample sizes of the two groups is 1 if the unilateral alpha is
0.05 and the beta is 0.2, the sample size of the two groups is calculated as 88
cases of the organoid-sensitive drug infusion group and 88 cases of the positive
control group. 88 cases and 88 cases in the positive control group, totalling 176
cases, and taking into account 20% shedding, a total sample size of 220 cases was
required.
3.7 Flow chart of research techniques This section (pictures) describes the technical
route of the project. 3.8 Data collection and management Data were collected by
observation and recording, and the original data were checked item by item to ensure that
the data were consistent with those in the original records. At the end of the
experiment, it was handed over to the statistician for statistical analysis.
3.9 Statistical analysis methods Measured data are generally descriptive statistics with
mean, standard deviation, median, minimum and maximum values. For categorical variables,
descriptive statistics will be performed according to frequency and percentage.
Statistical software SPSS was applied for statistical analysis. Measurement data were
expressed as mean ± standard deviation (x ± s), t-test, one-way ANOVA (post hoc test
two-by-two comparisons using the LSD method), chi-square test was used for comparison of
rates, and Spearman's method was used for correlation analyses. p < 0.05 was considered
that the difference was statistically significant.
3.10 Quality control 3.10.1 Class organ HE staining and immunohistochemistry results
Conform to the pathological test results, indicating that the constructed organoid and
tumour specimens can better retain pathological stability.
3.10.2 Organoid WES or RNA-Seq analysis Constructed organoid and tumour specimens can
better retain genetic stability. 3.10.3 Evaluation criteria for drug sensitivity test
results The evaluation standard of drug sensitivity test results has IC50,IR as the main
index, with the degree of deviation between IC50 and blood drug concentration, AUC
results as an auxiliary judgement, when the IC50, IR sensitive indexes are met at the
same time, it is regarded as sensitive; when it is not possible to meet the IC50, IR
indexes at the same time, IC50 combined with the IC50 and the need for the concentration
of the ratio of the smaller degree of difference in the results of the sensitivity.
3.10.3.1 IC50 evaluation criteria. IC50>20× Not sensitive
1×<IC50≤20× Low sensitivity 0.5×<IC50≤1× Moderately sensitive IC50≤0.5× highly sensitive
The sensitive group includes strongly sensitive and moderately sensitive, and the
insensitive group includes weakly sensitive and resistant.
3.10.3.2 Evaluation criteria for IR tumour inhibition rate . IR <30% is insensitive and
classified as negative; IR≥30% is sensitive and classified as positive, in which
30%≤IR<50% is low sensitivity, 50%≤IR<70% is moderate sensitivity, and IR≥70% is high
sensitivity.
Sensitivity rate = number of cases with IR ≥ 30% / total number of cases in the group.
3.10.3.3 Calculation of the degree of deviation of IC50 from blood concentration: the
degree of deviation from blood concentration refers to the degree of difference between
the IC50 value obtained from the drug sensitivity test and the blood concentration, and
is calculated as IC50/blood concentration.
The smaller the ratio, the more effective the drug. 3.10.3.4 Criteria for evaluation of
AUC (area under the drug-time curve) results: The specific calculation of AUC usually
involves integration of the concentration-time curve.
AUC is closely related to the dose-response relationship. In the dose-response curve of
drug efficacy, the AUC is a composite indicator of the overall effect of a drug on an
organism between dose and response. A larger AUC is usually associated with a better
therapeutic effect.
3.11 Post-study treatment of organoid The organoids constructed successfully in this
study will be uniformly destroyed and disposed of after the study in accordance with the
norms of biomedical waste disposal.
