Neoadjuvant Chemotherapy Combined With Preoperative Radiotherapy for Locally Advanced Breast Cancer
Phase: NA
Conditions: Breast Cancer
Sponsor: Cancer Institute and Hospital, Chinese Academy of Medical Sciences

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Research overview
Title: Prospective phase II clinical study of neoadjuvant chemotherapy combined
with preoperative radiotherapy in locally advanced breast cancer
Applicant: Shenzhen Hospital, Cancer Hospital, Chinese Academy of Medical
Sciences
Type of study: A clinical trial initiated by the investigator
Study subjects: Patients with locally advanced luminal (HER2-) and triple-negative
breast cancer
Objective: To prospectively evaluate the efficacy and safety of neoadjuvant
chemotherapy combined with preoperative large fraction radiotherapy followed by
surgery in breast cancer
Study design: A prospective phase II clinical study
Number of patients planned to be enrolled: 50
Clinical trial unit: Shenzhen Hospital, Cancer Hospital, Chinese Academy of Medical
Sciences
Radiotherapy: whole breast irradiation of the affected side ± chest wall + regional
lymph node drainage area irradiation: whole breast ± chest wall + supraclavicular
and internal mammary regional lymph node drainage area 40.05Gy/152.67Gy/ day
per session, axillary regional lymph node drainage area 37.5Gy/15f,2.5Gy/ per session
Primary endpoint: pCR rate (pathological complete response rate)
Secondary
safety
indicator
1:
incidence
of
acute
adverse
reactions
during
chemoradiotherapy (including acute skin injury, breast swelling and pain, local
infection, radiation mucositis, radiation pneumonia, upper limb lymphedema rate,
blood biochemical reaction and systemic reaction, etc.)
Secondary safety indicators 2: Postoperative complication incidence (delayed incision
healing rate, secondary surgery rate), late adverse reaction incidence (including late
skin injury, breast swelling and pain, cosmetic effect, radiation cardiomyopathy,

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pulmonary fibrosis, radiation brachial plexus injury, upper limb lymphedema), quality
of life (EORTC QLQ30, BR-23 questionnaire)
Secondary efficacy indicators: local recurrence rate, regional recurrence rate,
pathological response rate (according to Miller-Payne classification), npCR (near
npCR) rate, RCB (residual tumor burden) rate, event-free survival rate, distant
metastasis rate, tumor-free survival rate, and overall survival rate
Biological research: Collect fresh tumor tissue and peripheral blood before treatment
and surgery as test samples to evaluate biological markers such as tumor gene
expression, circulating tumor cells, inflammatory factors, ctDNA, etc.
Imaging studies: analysis of breast ultrasound and breast MR imaging parameters
before and after treatment.
Follow-up time: 5 years
Statistical methods: Based on the principle of intention processing analysis, pCR rate
and incidence of adverse reactions were calculated. Kaplan-Meier was used to
calculate local recurrence rate, regional recurrence rate, distant metastasis rate,
tumor-free survival rate and total survival rate.
technology roadmap ：
2 cycles of neoadjuvant chemotherapy followed by reduction of primary lesion
volume less than 50% + 3 weeks of neoadjuvant radiotherapy + 4 cycles of
neoadjuvant chemotherapy + surgery ± adjuvant therapy
1. Research Background
Breast cancer (BC) is the leading malignancy among women globally, ranking
first in cancer-related deaths and fifth in overall cancer mortality. In 2020, it
surpassed lung cancer to become the world's most common malignancy. China
accounts for 416,000 new cases annually, making it the primary cause of
cancer-related deaths among women and representing 18.4% of the global breast

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cancer burden.1 Non-metastatic locally advanced breast cancer (LABC) patients in
China account for about 12-15%, significantly higher than that in the United States
and Europe (about 8.5% in the United States and 4% in Europe).Due to its lower
overall survival rate, higher recurrence rate, and risk of distant metastasis, Localized
Axillary Breast Cancer (LABC) significantly impacts the overall efficacy and survival
of breast cancer patients. The current treatment model for LABC patients remains a
major clinical challenge. In clinical practice, these patients typically undergo primary
systemic therapy (PST) to achieve tumor downstaging before surgery, followed by
adjuvant treatments (primarily radiotherapy and medication). Radiotherapy serves as a
crucial therapeutic approach to reduce local recurrence rates and improve overall
survival rates.
Preoperative neoadjuvant radiotherapy (NART) is a common treatment option
for many tumors (such as esophageal carcinoma, rectal carcinoma, soft tissue sarcoma,
etc.). By bringing radiotherapy and systemic chemotherapy to the preoperative stage
to strengthen preoperative treatment, reduce the stage and eliminate micro-metastatic
lesions early to improve overall survival.3. In breast cancer patients, NART has the
opportunity to further shrink the tumor, improve the probability of breast preservation,
and may not need to perform axillary lymph node dissection, allowing axillary
surgery downgrading.4 Many studies have found that patients with pCR after
neoadjuvant therapy (NAT) generally have a better prognosis than non-pCR, and
NART is also one of the treatment options to reduce the stage.But this combination
has not yet been widely used in breast cancer patients.
In all breast cancers, luminal breast cancer accounts for 50-70% (LuminalA
type about 30-40%, LuminalB type about 20-30%), among which HR+HER2-type
breast cancer patients have poor efficacy of neoadjuvant chemotherapy, with
objective response rate (ORR) about 40-60%, partial response rate (PR) about 30-50%,
and complete response rate (CR) usually not more than 15%.When neoadjuvant
chemotherapy shows poor efficacy, these patients typically opt for direct surgical

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intervention followed by adjuvant radiotherapy and endocrine therapy. Research has
demonstrated that postoperative adjuvant chemotherapy can still improve prognosis in
axillary lymph node-positive LuminalA breast cancer patients. Therefore, even with
suboptimal short-term response rates, neoadjuvant therapy may still offer significant
benefits to the prognosis of axillary lymph node-positive Luminal breast cancer
patients.In terms of drug therapy, chemotherapy remains the primary treatment
approach, though options remain limited. Conventional neoadjuvant therapies such as
anthracycline, taxane, and platinum-based regimens have reached their therapeutic
limits, with pathological complete response (pCR) rates hovering around 35%. For
triple-negative breast cancer patients, studies have shown that improved pCR rates
translate to enhanced event-free survival (EFS) and overall survival (OS). Recent
years have witnessed active exploration of neoadjuvant therapies targeting
triple-negative breast cancer (TNBC) to achieve therapeutic breakthroughs. Current
research indicates that while chemotherapy combined with immunotherapy can
improve pCR rates and short-term survival advantages, the drug combination also
brings significant toxic side effects including mortality, potentially preventing many
patients from completing their planned treatment regimens (up to 10-20%). The
long-term tolerance of certain immunotherapy-related adverse reactions still requires
extended clinical follow-up, while the rising economic burden remains an
unavoidable challenge.In LABC, most patients require both chemotherapy and
radiotherapy, and the optimal delivery sequence is not known.In conclusion, for
HR+/HER2-triple-negative LABC patients with poor response to neoadjuvant
chemotherapy, radiotherapy can be considered as a precursor to improve the
probability of conversion to operable state, breast preservation and pCR.7–10
Poleszczuk et al. analyzed breast cancer patients in the SEER database and
found that preoperative radiotherapy was safe and effective, associated with increased
disease-free survival (DFS). The study also revealed that preoperative radiotherapy
reduced the incidence of secondary primary tumors compared to postoperative
radiotherapy. The authors suggested that neoadjuvant radiotherapy (NART) could

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directly target tumors for precision treatment, thereby activating specific immune
responses against cancer cells. This radiation-induced immunity not only helps
eliminate primary tumors but also removes microscopic lesions in both ipsilateral and
contralateral breasts, while reducing the risk of distant metastasis—effects absent in
postoperative radiotherapy. Additionally, neoadjuvant radiotherapy allows for
preservation of primary breast tumors, making tumor localization easier and
improving target delineation consistency. The preoperative APBI (Preoperative
Prostate Biopsy Imaging) and postoperative APBI (Postoperative Prostate Biopsy
Imaging) trials enrolled patients with similar tumor sizes, but the former demonstrated
significantly smaller target volumes (average PTV: 122cm³ vs. 296 cm³). The
reduced tumor target volume also minimized radiation-induced damage to normal
tissues.13. In addition, the hypoxia caused by surgical injury to the blood supply of
tumor tissue may increase the resistance of tumor cells to radiotherapy, resulting in
weakened anti-tumor effect and reduced efficacy of postoperative radiotherapy.In
addition to causing damage to BC tissues and cells, NART can also affect normal
tissues exposed to radiation 14.The results showed that tumors in the irradiated area
grew faster, while those in the non-irradiated area were halved in size. The authors
believe that this study altered the cellular or molecular biology of normal tissues
through preoperative radiotherapy, making the local environment unsuitable for
cancer cell proliferation and thus reducing the likelihood of tumor recurrence and
metastasis15.
Current clinical research shows insufficient exploration in the NART field. One
reason may be that NART may cause excessive fibrosis in tumor tissues and blood
vessels, complicating intraoperative tumor dissection. Clinicians generally believe this
could lead to more surgical complications. Additionally, balancing the timing between
radiotherapy and surgery poses challenges—safety must be ensured while minimizing
risks of tumor regrowth and metastasis, along with sufficient time for pathological
shrinkage and staging reduction. Therefore, strict control over the target area, dose,
duration of NART, and intervals between radiotherapy and surgery is essential.

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Studies have found that immediate surgery or intervals exceeding three months may
increase wound healing issues and postoperative complications. Since potential
fibrosis may complicate surgery and postoperative care, excessively extending
intervals between radiotherapy and surgery could heighten metastasis risks and delay
adjuvant therapy implementation, thereby reducing tumor recurrence likelihood. Most
reported intervals between NART and surgery range from 6 to 12 weeks.According to
the study of Chidley et al., NART can help to initially reduce tumor volume, thus
improving postoperative aesthetic effect.For patients, breast reconstruction surgery
can be performed at any time without causing tissue loss. From a oncology
perspective, it also avoids delays in postoperative radiotherapy due to complications
or prolonged recovery. Moreover, achieving overall target dose coverage becomes
easier without reconstruction implants.16
In terms of dose fractionation, clinical studies have shown that the α/β value
of breast cancer is lower than that of general tumors. Yarnold et al. calculated that the
α/β value of breast cancer sensitivity to a single dose was about 4.1Gy, which was
similar to 3.6Gy for advanced normal tissues.17 After 10 years of results from the
START study, the meta-analysis of RMH/GOC and START A studies provided
adjusted α/β values of 3.5Gy for locally recurrent breast cancer tissue and 3.1Gy
for normal reactive breast tissue.
Since the 1990s, randomized clinical trials including RMH/GOC, START A,
START B, Ontario Canada, DBCG HYPO, MDACC, and China Multicenter studies
have demonstrated that large-field radiotherapy (LFR) following breast-conserving
surgery is as effective as conventional fractionated radiotherapy without increasing
adverse events. These approaches not only reduce treatment costs and medical
resource consumption but also improve patients' quality of life. Notably, among 864
participants (14.7%) in the START series and 28 cases (3.8%) in the China
Multicenter study receiving regional lymph node irradiation, LFR was found to cause
no additional upper limb edema or shoulder joint dysfunction.Both the 2020 St Gallen

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International Guidelines and the 2022 European Society of Radiation Oncology
consensus recommend that regional lymph node irradiation can be performed with
large fractionation.Research indicates that whole breast fractionated radiotherapy
(WMFRT) delivers cosmetic outcomes comparable to conventional fractionated
radiotherapy without increasing breast-related adverse reactions. Studies further
demonstrate that WMFRT significantly improves the excellent-to-excellent cosmetic
response rate and reduces the incidence of grade 2 or higher acute radiation dermatitis.
The RMH/GOC and START studies revealed that late-stage adverse reactions in
patients receiving 39-40Gy of WMFRT were lower than those in conventional
fractionated radiotherapy groups, which may be attributed to the reduced equivalent
dose (EQD2=46 Gy) in WMFRT regimens.Yarnold18 et al. found that compared with
the dose effect curve of local control of breast cancer tumors, the dose effect curve of
normal breast tissue was steeper. The EQD2 of whole breast radiotherapy decreased
from 50Gy to 46Gy, and the local recurrence rate of tumor increased by ≤1.5%, but
the incidence of moderate and severe adverse reactions could be reduced by 30%.19
Combined regional lymph node irradiation (supraclavicular ± axillary lymph
drainage area) increases the incidence of upper limb lymphedema compared to simple
breast radiation therapy. In the START series study involving 864 patients receiving
regional lymph node irradiation, only one case of brachial plexus injury occurred.
Moreover, there was no significant difference in the incidence of upper limb
lymphedema between the large-field radiotherapy group and conventional
fractionation group (with similar rates at 5-year and 10-year follow-up). A Chinese
study on modified radical postoperative large-field radiotherapy for stage III disease,
which included chest wall and regional lymph nodes, also showed that large-field
radiotherapy did not significantly increase the incidence of upper limb lymphedema
compared to conventional fractionation—20.2% versus 20.5%, respectively.The
Korean study reported by 23Byun et al showed that the incidence of upper limb
lymphedema in the three-year period was significantly lower in the large fractionation
group than in the conventional fractionation group (6.8% vs. 13.5%), which may be

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related to the lower EQD2 (45-46Gy) in the large fractionation group.In 24 studies on
large-field radiotherapy after breast-conserving surgery and modified radical
mastectomy in China, no patients developed brachial plexus injury. Leong et al.
analyzed 708 patients across two centers, revealing that medium-dose large-field
radiotherapy did not increase upper limb dysfunction or brachial plexus injury
compared to conventional fractionation, while showing lower incidence of shoulder
joint dysfunction in the large-field group. All these studies employed medium-dose
large-field radiotherapy with a single dose <3Gy.25Johansson et al. reported that the
incidence of brachial plexus injury was almost zero when EQD2 was less than 57Gy,
so regional lymph node prophylactic irradiation with an appropriate dose of medium
large fraction theoretically would not increase brachial plexus injury.26 Studies
reported that the risk of lymphedema was significantly higher with axillary lymph
node dissection than with sentinel lymph node biopsy + regional lymph node
irradiation (supraclavicular and axillary I-III groups).In a prospective randomized
controlled phase 2 study published in JAMA by Schaverien et al., comparing
preoperative large-field radiotherapy (LFR) with conventional fractionated
radiotherapy, 50 breast cancer patients (25 each group) were enrolled in a 1:1
randomized trial. The treatment regimen included whole breast irradiation and
regional lymph node dissection (supraclavicular, internal mammary, and axillary).
Among these 45 patients, 60 underwent axillary lymph node dissection. The LFR
group received 40.05Gy/15f radiation for the entire breast and 37.5Gy/15 sessions for
regional lymph nodes. Notably, 6% (13%) of all patients developed clinical
lymphedema 8.3 months post-radiation completion.A total of 29 patients developed
Grade 1 CTCAE dermatitis, with 10 cases progressing to Grade 2 dermatitis upon
completion of radiotherapy (1 case in the large-field group versus 9 in the
conventional-field group; P=0.02). No patients experienced Grade 3 or higher
radiation-related toxicities or required treatment discontinuation. The study indicates
that the risk of lymphedema following axillary dissection after preoperative axillary
lymph node drainage with large-field radiotherapy remains safe and manageable for

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breast cancer patients. However, further evidence and extended follow-up periods are
needed to confirm these findings.
Current research remains inconclusive regarding the indications for internal
mammary radiation therapy (IMRT). Intraoperative IMR lymph node biopsy studies
indicate that the positive rate of internal mammary lymph nodes varies between 10%
to 40% depending on the tumor's quadrant, while the recurrence rate after systemic
treatment is less than 5%. Recent prospective multicenter postoperative adjuvant
radiotherapy studies have incorporated the internal mammary field into treatment
targets, demonstrating local control and long-term survival benefits. Guidelines
recommend IMR irradiation for patients with high preoperative imaging-predicted
internal mammary lymph node metastasis risk, primary tumors in the medial quadrant
or central region with axillary lymph node metastasis, or other high-risk internal
mammary lymph node metastasis probabilities. Notably, for patients with tumors in
the internal quadrant or central region and N2 status, the internal mammary lymph
node metastasis rate can exceed 40%.This study aims to perform intraluminal
radiotherapy in the inner quadrant/central region under full informed consent of
patients.
Currently, there are no definitive biomarkers in clinical practice to predict
whether breast cancer patients will benefit from radiotherapy. In translational research,
NART can directly evaluate the effectiveness of radiotherapy for breast tumors,
identify predictors and biomarkers of radiation response, advance radiobiological
studies of tumors and normal tissues, and guide personalized treatment plans for
breast cancer patients. A secondary objective of the PAPBI13 trial was to explore
gene expression profiles that predict radio-sensitivity. Significant changes in gene
expression were observed in specimens before and after radiotherapy, primarily
involving p53 signaling, cell cycle regulation, DNA repair, and inflammatory
responses. However, no clinically significant differences in gene expression were
found between patients who received radiotherapy and those who did not.Tanic et al.
identified differential expression of APOA1, MAP3K4, and MMP14 genes between
tumors with and without NRT response. Mitogen-activated protein kinase kinase 4

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(MAP3K4), a mitogen-activated protein kinase, was identified as a
radiotherapy-specific biomarker for predicting survival in patients with adjuvant
radiotherapy without distant metastasis. These findings suggest that MAP3K4 may
serve as a biomarker for predicting preoperative and postoperative radiotherapy
responses, as well as a potential target for combined radiotherapy-sensitizing therapy.
This suggests the need for further preclinical studies and prospective clinical trials to
validate these findings.The limitations of most studies lie in the selection of
biomarkers not based on their radiotherapy specificity, predominantly relying on cell
and animal models for foundational research, while clinical studies require large-scale
long-term follow-up. This study also contributes to organoid development: Organoids
are 3D cultured in vitro tissues that closely mimic cellular components, structural
features, and functional properties of in vivo tissues. Widely used in both basic and
translational research, they facilitate investigations into cancer onset, progression,
tumor heterogeneity, gene-drug interactions, target prediction, and drug sensitivity
testing. Compared with traditional 2D cell culture models, organoids exhibit complex
spatial configurations simulating differentiated tissues, demonstrating interactions
between cells and their surrounding matrix, as well as spatial positioning patterns.
They also exhibit physiological responses similar to in vivo differentiated organs and
maintain high similarity to their source tissues. These characteristics make organoids
highly promising for both basic research and clinical applications. The resulting
organoid disease models exhibit organ-specific structures and functions while
replicating in vivo disease manifestations, effectively avoiding species-specific
variations. Patient-derived tumor organoids (PDTOs) refer to models established
through in vitro cultivation of patients 'tumor tissues (typically malignant specimens
obtained via surgical resection or biopsy), which closely resemble the patient's tumor
type. Compared with other tumor models, PDTO (Patient-Derived Tumor
Oligonuclear Organoids) largely preserves the histological and genetic information of
original tumor tissues, maintaining structural similarity to the primary tumor. This
makes it an excellent in vitro "surrogate" for patient tumors. Additionally, tumor
organoids offer advantages such as ease of operation, minimal required tissue volume,

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rapid model establishment, convenient in vitro observation, and stable genome
stability. These features make them highly promising for disease modeling, clinical
cancer research, and drug sensitivity testing. With moderate costs, short culture
timelines, high success rates, and conversion rates, they are expected to provide a new
cost-effective platform for drug screening and development. Furthermore, continuous
monitoring of pre-and post-radiotherapy tumor biopsy specimens from patients with
known clinical responses can help evaluate radiobiological characteristics, specific
biomarkers/imaging markers, and optimal radiotherapy-drug combination regimens
for breast tumors/normal tissues. This approach promotes more effective treatment
selection, avoids overtreatment, and reduces associated medical expenses.4,30–32
In imaging evaluation, conventional breast X-ray and two-dimensional grayscale
ultrasound primarily assess neoadjuvant therapy efficacy based on tumor morphology
and size changes. However, relying solely on morphological and dimensional
measurements may fail to comprehensively evaluate neoadjuvant treatment outcomes,
as some tumors may show minimal volume changes while exhibiting diminished or
lost cellular activity. Compared to other breast imaging modalities, MRI demonstrates
advantages in multi-parameter, multi-slice functional imaging capabilities. Its
dynamic contrast enhancement, diffusion-weighted imaging (DWI), and magnetic
resonance spectroscopy (MRS) allow comprehensive assessment of tumor
morphology, hemodynamic changes, and functional metabolic alterations. Studies
confirm that breast MRI shows high consistency with histopathological evaluation of
neoadjuvant therapy efficacy in breast cancer, closely approximating the extent of
pathological changes compared to histological findings. Additionally, MRI
outperforms clinical palpation, X-ray, and ultrasound in distinguishing residual
lesions from fibrosis and detecting microscopic residual lesions post-neoadjuvant
therapy. As a standard imaging method, MRI has become the primary assessment tool
for evaluating breast cancer's response to neoadjuvant therapy. Conventional breast
MRI evaluation metrics include tumor morphology and size, time-signal intensity
curve patterns after dynamic contrast enhancement, MRI
semi-quantitative/quantitative parameters, ADC values from DWI, and cholinergic

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peak variations from MRS. While non-invasive tumor prognosis assessment and
predictive biomarkers hold significant advantages, their clinical validation and
application progress remain slow. Preoperative treatment studies demonstrate
advantages in validating imaging biomarkers, enabling correlation analysis between
preoperative/interoperative imaging features and pathological/molecular endpoint
indicators. Advances in imaging-based tumor biology capabilities could, in turn,
facilitate research into adaptive radiotherapy using strategies such as dose-fine
distribution, individualized dose and fractionation plans, and radiotherapy combined
with drugs.4,33,34
The evolving multidisciplinary management paradigm for breast cancer has
driven our efforts to develop personalized treatment strategies and integrate various
approaches. As a non-mainstream treatment modality, preoperative high-dose
neoadjuvant radiotherapy (NART) has emerged as a novel therapeutic option for
locally advanced luminal (HER2-)-and triple-negative breast cancer patients who
have demonstrated suboptimal response to neoadjuvant chemotherapy. This study
aims to investigate the efficacy and safety of combining neoadjuvant chemotherapy
with preoperative high-dose radiotherapy in locally advanced luminal (HER2-)-and
triple-negative breast cancer patients through a prospective Phase II single-arm trial.

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2. Objective of the study
2.1 Research task: Prospective enrollment of eligible patients with locally
advanced breast cancer, using intensity-modulated technology to implement
preoperative large fraction of whole breast ± chest wall + supraclavicular lymph
node drainage area + axillary lymph node drainage area ± internal mammary lymph
node drainage area. Follow-up observation of radiation toxicity and efficacy.
2.2 Research objectives: To clarify the safety and feasibility of preoperative large
fraction radiotherapy for patients with locally advanced breast cancer, and to provide
a basis for future phase III clinical studies.
2.1
Objectives:
Eligible
patients
with
locally
advanced
breast
cancer
were
prospectively enrolled. Imrt was used to perform preoperative hypofractionated whole
breast irradiation ± chest wall + supraclavicular lymph node irradiation+axillary
lymph node irradiation± internal mammary lymph node irradiation. The toxicity and
efficacy of radiotherapy were followed up.
3. Study endpoints
3.1 Primary study endpoint: pCR rate (pathological complete response rate)
3.2 Secondary study endpoints:
3.2.1 Early adverse reactions: acute skin injury, breast swelling and pain, local
infection, radiation mucositis, radiation pneumonia, upper limb lymphedema rate,
blood biochemical reaction and systemic reaction
3.2.2 Other adverse events:
Postoperative complication rate (delayed incision healing rate, secondary surgery rate),
late adverse reaction rate (including late skin injury, breast swelling and pain,
cosmetic effect, radiation cardiomyopathy, pulmonary fibrosis, radiation brachial
plexus injury, upper limb lymphedema), quality of life (EORTC QLQ30, BR-23
questionnaire)
3.2.3 Efficacy indicators: local recurrence rate, regional recurrence rate, pathological
response rate (according to Miller-Payne classification), npCR (near npCR) rate, RCB
(residual tumor burden) rate, event-free survival, distant metastasis rate, tumor-free

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survival rate, and overall survival rate
3.2.4 Transformation research indicators:
Tissue samples: including pre-adjuvant therapy and surgical specimens. Some are
used for organoid construction; others are used for single-cell sequencing; paraffin
tissue samples: some tissue blocks are fixed in centrifuge tubes containing formalin,
which can be used for spatial transcriptome sequencing.
Peripheral blood samples: The plasma part can detect immune factors and ctDNA,
and PBMC (peripheral blood mononuclear cells) can be enriched by flow or mass
spectrometry, as well as T and NK. CTDNA is planned to collect 5-10 patients.
（1）Fecal Sample Collection Protocol: Collect stool samples at the following
intervals: 1 week prior to radiotherapy, within 3 days after radiotherapy completion, 1
week before initial chemotherapy, 1 week before the 3rd and 5th chemotherapy cycles,
1 week preoperatively, and 1 week postoperatively. (Pre-collection instructions:
Record the patient's oral antibiotic or probiotic intake history from 2 weeks prior to
the designated collection time. Instruct patients to collect stool in a clean container
(e.g., disposable toilet bowl), avoiding contamination from urine or toilet surfaces.
Collect fresh mid-to-late portion stool using a sterile spoon inside the collection tube
(1-3 scoops, 1g). Store samples in-80℃°C freezer within 30 minutes after collection.
Sample labeling: Tumor type code + medical record number + collection date. If
possible, collect family members' samples as paired control specimens.)
（2）Saliva: Samples are collected one week before radiotherapy, within three
days after radiotherapy completion, one week before the first chemotherapy session,
one week before the 3rd and 5th chemotherapy cycles, and one week before surgery.
(Pre-collection instructions: Record the patient's history of oral antibiotics, probiotics,
and dental procedures (like fillings or extractions) two weeks prior to the designated
time point. Ensure the patient rinses their mouth with 30ml of purified water for 30
seconds on the collection day, then empties it into the saliva sampling cup. Store
samples in an-80℃freezer within 30 minutes after collection. Document detailed
sampling times (Sample code: Tumor type code + Medical record number +
Collection time). If possible, collect family members as paired control samples.)

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（3）Imaging: Breast MR was performed 2 weeks before radiotherapy, 1 week
before the first chemotherapy, 1 week before the 3rd and 5th cycle of chemotherapy,
and 1 week before surgery
（4）Imaging: Breast MR was performed 2 weeks before radiotherapy, 1 week
before the first chemotherapy, 1 week before the 3rd and 5th cycle of chemotherapy,
and 1 week before surgery
（5）Imaging part: MRI of breast was performed 2 weeks before radiotherapy, 1
week before the first chemotherapy, 1 week before the third and fifth cycles of
chemotherapy, and 1 week before surgery
4. Enrollment screening criteria
Entry criteria
Women aged 18 to 70
1) The pathological diagnosis was invasive breast cancer
2) cT3-4, cN1-2, M0 (AJCC Cancer Staging Manual, 8th edition), molecular
subtype was luminal (HER2-) and triple-negative, and the primary lesion
volume was reduced by less than 50% after 2 cycles of neoadjuvant
chemotherapy
3) Generally in good condition (KPS>70 points), organ function is tolerable, and no
severe complications of poor control
4) Generally in good condition (KPS>70 points), organ function is tolerable, and no
severe complications of poor control
5) MRI scans are acceptable
6) Be eligible for follow-up
7) Sign the informed consent form
Exclusion criteria
1)
gestation period
2)
Simultaneous bilateral breast cancer
3)
History of chest radiotherapy
4)
There is active connective tissue disease
5)
Inflammatory breast cancer

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6)
Breast size is large (>600ml) and BMI>30
7)
diabetes mellitus
8)
The upper and lower regions of the clavicle and the internal mammary region
showed positive lymph nodes
9)
Use of immunotherapy drugs such as PD-1/PD-L1
10) History of other malignancies, excluding but not limited to curable early papillary
thyroid carcinoma, non-malignant melanoma skin cancer, cervical carcinoma in
situ, contralateral breast carcinoma in situ, early lung cancer, etc
Standards for shedding
1）Significant tumor progression during radiotherapy or systemic therapy
2）Serious non-oncological disease that makes continued treatment or routine
follow-up impossible
3）The patient volunteered to withdraw
Baseline check, Table 1
Pre-entry screening (mandatory)
testing time
History and physical
examination
General condition score, height, weight
Within 1 week prior to treatment
Pathological review
Postoperative pathological sections from the external
hospital (lower level hospital) need to be reviewed again
by the research unit, including relevant
immunohistochemistry
Pre-treatment preparations
routine blood test
Absolute white blood cell, lymphocyte and neutrophil
count, hemoglobin, platelets
Within 1 week prior to
treatment
Liver and kidney function
AST, ALT, total bilirubin, creatinine
blood fat
Triglycerides (TG), total cholesterol (TC), low density
lipoprotein cholesterol (LDL-C)
myocardial enzyme
CK-MB,TNI, NT-proBNP

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iconography
Breast X-ray/ breast B-ultrasound + breast
MRI;
Chest enhanced CT (preferably thin layer)
in our hospital;
B-ultrasound of bilateral axilla, neck, supraclavicular and
inner breast;
Liver ultrasound/or enhanced CT/or MRI;
brain MR；
Whole-body bone scan
Whole-body PETCT (optional)
Within 2 weeks (maximum 4
weeks) prior
to whole body
treatment
Toxicological evaluation
Baseline toxicity assessment, photos (beauty
evaluation), and written records
1 week before radiotherapy
Table of breast beauty
effect and quality of life
BREAST-Q questionnaire
Thyroid examination
T3, T4, FT3, FT4, TSH; thyroid ultrasound
Within 1 month before
treatment, after every 2 cycles
of chemotherapy,
preoperatively, and 3 months,
6 months, and 1 year after
surgery
cardio-pulmonary
function
Twelve lead electrocardiogram
echocardiogram
lung function
Two weeks before surgery
(maximum 1 month)
Organizational specimens,
blood specimens, urine
and stool specimens
Fine needle aspiration tissue and surgical specimens
before radiotherapy
Blood specimens and urine specimens were collected
within 2 weeks before radiotherapy, on the day of
radiotherapy end, before the first, third and fifth cycles of
chemotherapy, preoperatively, postoperatively, 3 months
after surgery and 1 year after surgery
5. Experimental design and statistical processing

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5.1. Sample size
This exploratory study investigates the design and optimization of neoadjuvant
chemotherapy combined with preoperative medium-dose fractionated radiotherapy
(MFRT) in breast cancer. The prospective single-arm Phase II clinical trial aims to
compare pathological complete response rates (PBR) and acute-phase adverse event
(AEE) rates between this regimen and standard treatment protocols. Specifically, it
evaluates the efficacy of preoperative MFRT in locally advanced luminal
HER2-negative triple-negative breast cancer patients. Given the exploratory nature of
the study and limited prior research on preoperative MFRT, a total sample size of 50
cases was determined to ensure robust analysis.
5.2 Statistical analysis
1)
Kaplan-Meier statistical analysis was used for survival analysis, log-rank test was
used to compare survival curves, and Pearson's chi-square test was used to
compare categorical variables. When the p value was less than 0.05, the
difference was considered statistically significant.
6. Radiation therapy
Prior to initiating treatment, the primary tumor site should be marked with
photographic documentation for subsequent resection. The primary lesion may be
identified using tattoos, superficial projections, or metallic markers. These metallic
markers can be placed at the tumor center and/or margins. When no intramammary
markings are available, external identifiers such as superficial tattoos, mass
projections, or diagrams may be employed. Clinical examinations combined with
ultrasound imaging should thoroughly evaluate axillary lymph nodes. If ultrasound
suggests suspicious axillary lymph nodes during baseline assessment, a guided
ultrasound-assisted biopsy should be performed to determine the status of these
lymph nodes.
Photography (front, left oblique, right oblique): 1 week before radiotherapy, the day

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of radiotherapy end, 1 week before surgery, 1 month after surgery
Radiotherapy: CT positioning is used to delineate the target area and normal tissue
for dose assessment.
6.1 Simulation positioning
CT positioning: The patient is placed in a supine position with the affected upper
limb abducted to grasp the rod. Positioning fixation is performed using a breast
support frame combined with a headplate or a cervical-thoracic integrated frame
with cervical-thoracic membrane (TOMO treatment requires prolonged duration and
necessitates the use of a cervical-thoracic integrated frame with membrane fixation).
A plain CT scan is performed, with the scanning range extending from below the
jawline at the upper boundary to encompass the entire liver at the lower boundary,
using 5mm layer thickness and lead wire markers for the entire breast area
6.2 Exposure range
Target delineation:
CTV: The whole breast on the affected side, up to 5mm subcutaneously
PTV: CTV extends 5mm outward and retracts to 5mm subcutaneously
CTVsc: Suprascapular lymph node drainage area
CTVax: Axillary lymph node drainage area
CTVim: Internal mammary lymph node drainage area
PTVsc/ax/im: CTVsc/ax/im were expanded 0.5cm outward in three dimensions
and retracted 0.5cm subcutaneously
Usage of bolus: Patients with skin invasion used 0.5cm thick bolus throughout
the whole course to improve the dose on the skin surface, and the size was 2cm wider
than the affected skin.
Table 2. Reference anatomical boundaries for CTV delineation of the whole
breast, chest wall, above and below clavicle, axilla, and inner breast
upper
the world of
mental
the external
prezong
The backside

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bound
mortals
state
world
mammary
gland
Clinical
border /
level of the
second
anterior rib
Clinical
border/CT
seen
disappearance
of breast
layer
The
junction of
the ribs and
sternum
Breast
folds/adenoids
seen on CT
Under 5mm of skin
Excluding
the pectoral
muscles,
chest wall
muscles and
ribs
walls of
the chest
Lock the
lower edge
of the bone
horizontally
Breast
underscoring
level
Within
1cm of the
midline
midaxillary
line
The posterior
boundary of the
breast
Posterior
border of
pectoralis
minor
Above the
collarbone
Lower edge
of the
annular
cartilage
0.5cm below
the lower
margin of the
subclavian
vein
The inner
edge of the
internal
carotid
artery or
vein
Medial border
of trapezius
muscle,
medial border
of pectoralis
minor
Superficial to the
sternocleidomastoid
muscle and deep to
the platysma
The ventral
surface of the
scalene
muscle
Axillary
Group III
(subclavicular
area)
Start level
of
pectoralis
minor
0.5cm below
the axillary
vein in the
medial border
of the
pectoralis
minor muscle
The junction
of the
subclavian
vein and the
jugular vein
Medial border
of pectoralis
minor
Posterior border of
pectoralis major
Superior
5mm of the
subclavian
vein or
muscle at the
rib/intercostal
space
Axillary I
group
The
axillary
vein runs
along the
The
pectoralis
major fused
with the ribs
Medial
border of
pectoralis
minor
The medial
border of the
latissimus
dorsi
The anterior border
of the pectoralis
major or latissimus
dorsi muscle
The anterior
border of the
subscapular
muscle

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lateral
border of
the
pectoralis
minor
muscle
Axillary II
group
The
axillary
vein runs
along the
medial
border of
the
pectoralis
minor
muscle
The axillary
vein runs
along the
lateral border
of the
pectoralis
minor muscle
Medial
border of
pectoralis
minor
Medial border
of pectoralis
minor
The anterior border
of the pectoralis
minor
The ribs or
intercostal
muscles
endosperm
0.5cm
below the
lower
margin of
the
subclavian
vein
The upper
edge of the
fourth
anterior rib
cartilage
Intrauterine
blood
vessels are
0.5cm
0.5cm inside
the breast and
outside the
blood vessel
0.5cm anterior to
the internal
mammary vein
(resection at the
chest wall)
0.5cm
posterior to
the internal
mammary
vein
(resection at
the lung
tissue site)
Normal: Threatened organs: contralateral breast, ipsilateral and contralateral lungs,
heart, coronary arteries (left anterior descending artery LAD, right coronary artery
RA), affected shoulder joint, esophagus, thyroid gland, brachial plexus, liver, stomach,
spinal cord (spinal cord PRV)
Normal tissue delineation:

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Heart (heart): Upper boundary: the lower edge of the right pulmonary artery trunk,
lower boundary to the heart tip, corresponding level including the pericardium.
Left anterior descending artery (LAD): The LAD originates from the aortic root
and extends as the left main trunk of the coronary artery, approximately 2cm long.
It then branches into the LAD, which runs along the interventricular groove. The
LAD does not include the left main trunk and descends to outline the entire
length up to the cardiac apex. Considering the PRV caused by cardiac pulsation,
the LAD is outlined with a uniform diameter of 1cm.
Right main coronary artery (RA): Originates from the aortic origin, runs along the
right atrioventricular septum, and delineates the full length. Considering the PRV
and LAD of the heart beat, delineate a uniform diameter of 1cm.
Left and right lungs (lung L, lung R): The lung window is drawn separately, and
the left and right lungs are drawn separately. It includes the small blood vessels
extending into the lung tissue from the hilum, but does not include the
mediastinum structures such as the hilum, trachea and bronchus.
Spinal cord (cord): delineates the boundary of the spinal cord. 5mm is expanded
in front, back, left and right to form the spinal cord PRV (cord PRV). The upper
boundary is to the base of the skull and the lower boundary is to the lower edge of
the second lumbar vertebra.
The affected shoulder joint (L/R): outlined on the bone window, including the
entire humeral head and the joint cavity and articular surface
Affected brachial plexus (L/R): According to the RTOG delineation guidelines,
the brachial plexus is delineated from C4-5 to 1cm below the olecranon bone, and
the brachial plexus is delineated from the intervertebral foramen to the anterior
and middle scalene muscle space, with a uniform diameter of 0.6cm.
Thyroid (thyroid): outline the left and right lobes and isthmus of the thyroid
gland.
Esophagus (esophagus): from the entrance of the esophagus to the lower border
of the aortic arch.
Liver: The whole liver is required.

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The stomach (stomach) is required to be delineated at 2cm below the PTV.
6.3 Radiation therapy plan requirements
Table 3
target
section
Limitations
Lymph node drainage area of whole
breast/thoracic wall + supraclavicular
and internal breast area: 40.05Gy/15
times, 2.67Gy/time/day; lymph node
drainage
area
of
axillary
area:
37.5Gy/15f,2.5Gy/ times/day; both are
5 times per week and completed in 3
weeks;
Dosage
indicators
Limitations
ensemble PTV
（PTVsc/PT
Vax/PTVim/
PTV）
Prescription
dosage
target
D95%
40.05Gy
Offset is
acceptable
V40.05
90%
Hotspot
doses
objective
Dmax
<48Gy
Offset is
acceptable
V48
<1ml
Hotspot
doses
target
V44
<5%
Offset is
acceptable
V48
<1%
Whole body
Hotspot
target
Dmax
<48Gy

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Normal tissue limit:
normal
structure
Limitations
Lymph
node
drainage
area
of
whole
breast/corctaneous wall + supraclavicular
and internal mammary region: 40.05Gy/15
times,
2.67Gy/time/day;
lymph
node
drainage
area
of
axillary
region:
37.5Gy/15f,2.5Gy/ times/day; both are 5
times per week and completed in 3 weeks;
Dosage indicators
Limitations
Heart (left breast
cancer)
target
mean dose
<8Gy
Offset is
acceptable
<10Gy
objective
V5
<45%
Offset is
acceptable
<50%
organization
(Including
organized and
unorganized
sketches)
doses
Offset is
acceptable
V48
<1ml

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Heart
(right
breast cancer)
objective
mean dose
<5Gy
Offset is
acceptable
<6Gy
target
V5
<30%
Offset is
acceptable
<35%
LAD
objective
V40
<20%
Offset is
acceptable
<25%
RA
objective
V40
<20%
Offset is
acceptable
<25%
Lung on the
affected side
objective
mean dose
<15Gy
Offset is
acceptable
<16Gy
objective
V20
<30%
Offset is
acceptable
<32%
target
V5
<55%
Offset is
acceptable
<60%
Healthy
side of lung
target
V5
<20%
Offset is
acceptable
<25%

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Healthy breast
objective
mean dose
<5Gy
Offset is
acceptable
<8Gy
medulla
spinalis PRV
target
maximum dose
<30Gy
Offset is
acceptable
<32Gy
Inferior brachial
plexus injury
objective
maximum dose
<48Gy
oesophagus
objective
V30
<48Gy
Offset is
acceptable
<50Gy
The
shoulder
joint on the affected
side
target
V30
<30%
Offset is
acceptable
<35%
thyroid gland
objective
mean dose
<28Gy
Offset is
acceptable
<32Gy
Liver (right breast
cancer)
objective
V5
<25%
Offset is
acceptable
<30%
Stomach (left breast
cancer)
target
V5
<25%
Offset is
acceptable
<30%
Liver (left breast
target
V5
<10%

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cancer)
Offset is
acceptable
<15%
Stomach (right
breast cancer)
objective
V5
<10%
Offset is
acceptable
<15%
6.4 Irradiation dose
1）Whole breast ±
Thoracic wall + supraclavicular and internal mammary area
lymph node drainage area: 40.05Gy/15 times, 2.67Gy/time/day; axillary area lymph
node drainage area: 37.5Gy/15f,2.5Gy/ times/day;
2）If the primary lesion after surgery does not reach pCR, the tumor bed is increased
to 13.35Gy/2.67Gy/5F (the total EQD2 of tumor bed = 60Gy);
3）If the axilla is non-R0 resection, postoperative residual lesions should be
considered for additional treatment. Residual lymph nodes should be routinely
divided with 26Gy/13F after surgery, and the total radiation dose before and after
surgery should be 66Gy (EQD2)
6.5 Radiotherapy techniques
1) All of them adopt whole intensity modulation technology, which can be IMRT or
VMAT
2) It is recommended that all left breast cancer patients choose respiratory control
technology, and right breast cancer patients may consider using respiratory control
technology.
3) All plans must be physically QA validated before treatment can begin
4) Daily CBCT location verification is required during treatment
7. Systemic treatment
The chemotherapy regimen, endocrine therapy, and other pharmacological treatments
are standardized according to the latest domestic and international guidelines, along

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with recommendations from oncology specialists. The treatment course consists of six
cycles: two before radiotherapy and four after radiotherapy. If tumor shrinkage
remains below 50% after four cycles of chemotherapy, surgical intervention may be
considered. Endocrine therapy and other medications are initiated post-surgery.
8. Surgery
1)
Breast-conserving surgery is preferred whenever possible; autologous flap
transplantation may be considered if the patient strongly requests and accepts the
associated risks. The specific procedure is determined by the surgeon
2)
For
patients
with
trans-quadrant multifocal
tumors
at
diagnosis,
radical
mastectomy is recommended regardless of the response after neoadjuvant therapy;
if it is multifocal in the same quadrant, surgical mode can be selected according
to the surgeon's advice;
3)
If no positive lymph nodes are detected during preoperative axillary assessment,
sentinel axillary lymph node biopsy (SLND) may be performed first. For patients
with cN1 stage before neoadjuvant therapy, if clinical evaluation after
neoadjuvant therapy shows negative axillary lymph nodes and the biopsy
retrieves ≥3 sentinel lymph nodes with confirmed pathological negativity,
axillary lymph node dissection (ALND) may be considered as an option. If
metastasis is observed in any sentinel lymph node, ALND should be performed,
with specific procedures guided by surgical consultation.
9. Surgical pathology
9.1 The size of the primary lesion must be measured
9.2 Primary tumors must be graded according to the Nottingham revision of the
Bloom & Richardson grading system
9.3 The surgical margins must be assessed and reported
9.4 Routine immunohistochemical detection of ER, PR, Her2 and Ki67
9.5 Pathological response rate (must be classified according to Miller-Payne
classification), pCR was defined as: ypt0/Tis ypN0

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10. Onboarding process
10.1 The eligible patient shall sign the informed consent form
10.2 Complete radiotherapy as required
10.3 Fill in the CRF form
Any subsequent study-specific procedures or assessments may be performed after the
subject signs the informed consent form (ICF)
Screening process schedule:
Research phase
Screening period
flow path
Day 14 to Day 1 (D-14 to D-1)
Sign the informed consent form/assign subject number
X
Demographic data and medical history, smoking history,
alcohol consumption history
X
History of previous tumors and previous treatment
X
Inclusion/exclusion criteria
X
Clinical examination and evaluation
Physical examination, height, weight, blood pressure
X
vital sign
X
Chest, abdomen and pelvis CT (PETCT can be omitted if
complete)
X
Breast MRI, brain MRI
X
Whole body bone scan /PETCT
X
Cervical, thyroid and breast ultrasound
selectable
12 lead electrocardiogram
X
echocardiogram
X
ECOG physical score
X
Concomitant medication/treatment
X
Laboratory tests/assessments
Day 14 to Day 1 (D-14 to D-1)
Biopsy of breast mass, axillary lymph node biopsy
X
Pathological results
X

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Cancer markers CEA, CA15-3, CA-125
X
routine blood test
X
Blood biochemistry
X
Urinalysis, stool analysis
X
Cardiac markers
X
Thyroid function tests (TSH, free T3, free T4)
X
Coagulation function tests
X
Pregnancy test, serum βhCG
X
HBV, HCV, HIV virus screening
X
Tumor evaluation
Imaging examination (breast enhanced MRI + whole body
enhanced CT is preferred)
X
Tumor lesions were evaluated based on RECIST 1.1
X
Quality of life assessment
X
11. Efficacy evaluation
11.1 Long-term follow-up after surgery (see the follow-up table in Appendix for
details).
11.2 The follow-up form should be completed for each follow-up visit, and any
tumor recurrence, metastasis and death should be recorded on the follow-up form
11.3 Physical examination, breast and axillary, neck and supraclavicular ultrasound,
neck, chest and abdominal CT every 3-6 months for the first two years, bone scan,
cranial MR: routine once a year, timely review when relevant symptoms occur.
cCR criteria: Complete absence of enhancement in the primary tumor bed and
primary metastatic lymph nodes on MRI after NAC is defined as complete imaging
remission. In principle, surgical resection is still recommended for patients who
achieve cCR. If resection is not willing for any reason, MDT should be discussed to
make a decision.

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12. Assessment of acute and late radiotherapy side effects
12.1 Postoperative follow-up for 5 years
12.2 The follow-up form should be completed for each follow-up visit, and all
adverse reactions should be recorded on the follow-up form
12.3 The main adverse reactions include: swelling and pain of the breast on the
affected side, acute radiation dermatitis, radiation pneumonia, breast beauty effect,
radiation heart injury, radiation pulmonary fibrosis, lymphatic edema, brachial
plexus neuralgia
12.4 Adverse reactions were graded using the CTCAE 3.0 standard, and
lymphedema was assessed using the arm, shoulder and hand disability questionnaire
(QuickDASH-9, clinical lymphedema was defined as at least one reported relative
percentage difference of 10% or more compared to the baseline, healthy upper limb)
and the arm symptom subscale of cancer treatment function assessment-breast +4
(FACT-B+4)
12.5 Physical examination
12.6 Breast beauty evaluation (including physical examination and breast photos)
13. Quality of life assessment
13.1 Chinese version of EORTC QLQ-C30 scale and QLQ-BR23 scale
13.2 Assessments are performed before, after and at 1, 3, 6, 12,18 and 24 months
post-radiation therapy
Basic Requirements ：
1) The CRF should be completed for each follow-up visit, and all adverse reactions
should be recorded on the follow-up form.

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2) The evaluation criteria for acute and late radiotherapy adverse reactions were based
on CTCAE 3.0.
3) Upper limb edema was compared with the baseline and the measurement of the
circumference of the healthy upper arm. If the increase was more than 10%, edema
was considered to exist.
4) For the evaluation of skin injury and upper limb dysfunction, photos and videos are
preferred.
5) Adverse reactions occurring 3 months after the start of radiotherapy are considered
as late adverse reactions.

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