A Prospective, Single-Arm, Phase II Trial of Apatinib Combined With Nab-Paclitaxel and Carboplatin for The Neoadjuvant Treatment of Triple-Negative Breast Cancer

Background: Pathological complete response (pCR) is essential for improvements of prognosis in triple-negative breast cancer (TNBC). We evaluated the ecacy of apatinib combined with nab-paclitaxel and carboplatin in patients in this phase II clinical trial. Methods: Women with hormone receptor- and human epidermal growth factor receptor 2 (HER2)-negative, stage II/III breast cancer received six cycles of 75 mg/m2 docetaxel, carboplatin (AUC = 5) and 15 mg/kg bevacizumab every 21 days. The primary end point was pathological complete response (pCR) in the primary breast and axillary lymph nodes (ALN). Results: Thirty-two patients were recruited into the clinical trial, the vast majority of the patients had stage III tumors (65.6%) and the median longest tumor size was 3.5 cm. The pCR rate was 43.8% (n = 14); clinical response rate 93.8% (n = 30); complete response rate 21.9% (n = 7); partial response rate 71.9% (n = 23); stable disease 6.2% (n = 2). After surgery, 7 (63.6%) of the 11 patients without axillary lymph node metastasis achieved a pCR. The median target lesions in breast reduced to 1.2 cm after the third cycle treatment and 0.9cm after the last cycle. Most frequent grade 3/4 adverse events were thrombopenia (40.6%, n = 13) and neutropenia (25%, n = 8). Conclusions: Neoadjuvant apatinib, combined with albumin paclitaxel and carboplatin resulted in an encouraging pCR rate in locally advanced breast cancer and no major safety concerns during the therapy.

Conclusions: Neoadjuvant apatinib, combined with albumin paclitaxel and carboplatin resulted in an encouraging pCR rate in locally advanced breast cancer and no major safety concerns during the therapy.
Clinical trial registration: NCT03650738 Background Challenges were existed in treatment of triple-negative breast cancer (TNBC) owing to the short of appropriate targets (1). With the absence of hormone receptor and human epidermal growth factor receptor 2 (HER2), de nitive therapy such as antihormonal treatment and anti-HER2 monoclonal antibody were improper (2,3).
Aggressive nature and frequent visceral metastases of TNBC leads to the poor prognosis of patients, including short disease-free intervals (DFS), progression free survival (PFS) and overall survival (OS)(4-6). As a result, pathological complete response (pCR) after neoadjuvant therapy was pursued to achieve comparable survival outcomes in patients with TNBC (7,8). Currently, data from clinical studies support that neoadjuvant therapy plays an important role improving the pCR rates and long-term prognosis(8).
Clinically, only about one-third of stage II-III TNBC patients achieve pCR after anthracycline and taxanebased neoadjuvant chemotherapy (NAC) (9,10). More than half of TNBC patients still have invasive lesions remaining after NAC, which leads to clinical on the rapid metastasis and recurrence (7,8). Therefore, increasing the pCR rate after NAC is the top priority for improving the prognosis of TNBC patients, and it has also become an urgent clinical problem that needs to be resolved. Nab-paclitaxel: the rst chemotherapy drug based on a nanotechnology platform that does not require solvents, with active transport mechanism. After endocytosis, it is transported by endothelial cells and aggregated to the tumor site by albumin (4). At the same dose, the accumulation of drugs in tumor tissues after nab-paclitaxel administration is more e cient than paclitaxel, and shows better tumor/normal tissue selectivity (11,12). Several randomized controlled studies compared the e cacy and safety of nab-paclitaxel and paclitaxel/docetaxel, and the results suggested that the pCR rate of the TNBC subgroup was 41% (95% CI 38%-45%) (13,14). Compared with paclitaxel treatment, it can obtain higher pCR rate and signi cantly reduce the incidence of allergic reactions.
Most TNBCs exhibit a "BRCAness" phenotype due to BRCA1/2 gene mutations or other molecular changes (15,16), and usually have defects in the DNA repair mechanism, so they are particularly sensitive to DNA damaging agents. Platinum belongs to DNA damaging agent that kills tumor cells by inducing double-stranded DNA breaks (17). CALGB 40603(18) and GeparSixto (19) have shown that the pCR rate can be as high as 75% after the introduction of platinum in the neoadjuvant scheme. On the other hand, angiogenesis associated genes are frequently activated in TNBC, and inhibiting angiogenesis may be another strategy for TNBC treatment. The GBG44 and NSABP B-40 (20) study published in 2012 showed that the combined use of anti-angiogenesis therapy in neoadjuvant chemotherapy increased the pCR by about 4%-11%, opening up a new treatment for anti-vascular targeted therapy for TNBC. Apatinib, by highly selective competition for the ATP binding site of VEGFR-2 in cells, block downstream signal transduction and inhibit tumor tissue angiogenesis, have demonstrated good results in patients with advanced breast cancer in previous research (21,22), but currently lack experience in the application of neoadjuvant therapy.
Here, we performed a single-arm, randomized, phase II trial for patients with Stage IIb-IIIc breast cancer, aiming determining whether the addition of apatinib to neoadjuvant chemotherapy increase the rate of pCR.
Patients with major surgery within 4 weeks prior to enrollment or surgical wounds have not healed, or embolization and bleeding occurred within 4 weeks preceding enrolment, or malignant tumors of other histological origins in the past 5 years (except for cured cervical carcinoma in situ and basal cell carcinoma or squamous cell carcinoma) were ineligible. Severe cardiovascular disease including hypertension (BP ≥ 160/95mmHg) uncontrolled by medical treatment, unstable angina, history of myocardial infarction in the past 6 months, congestive heart failure > NYHA II, severe heart rhythm Abnormalities and pericardial effusions must be excluded, as were those suffering severe infection or mental illness, poor compliance.
This trial was executed in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki, as was approved by the Ethics Committee of the First A liated Hospital of Nanjing Medical University (2018-SR-115).
All the patient volunteers and signs an informed consent form (ClinicalTrials.gov identi er: NCT03650738).

Treatment
Patients received six cycles of 260 mg/m 2 albumin paclitaxel and carboplatin (AUC = 5-6) intravenously, once every 21 days, in combination with apatinib 250mg oral, continuous dosing. Patients with progressive disease by clinical or radiological con rmation or intolerable toxicity were withdrawn from the study. Regular follow-ups were conducted to evaluate safety and effectiveness. If the toxicity recovery does not meet the criteria for subsequent chemotherapy, the start of the subsequent cycle can be postponed appropriately, but the delay cannot exceed 14 days.

Study end points
The primary end point of the study was pCR rate, de ned as absence of invasive breast cancer in the primary breast and ALN. Secondary endpoints were toxicity.

Assessment
Tumour and axillary nodal status were evaluated as baseline, and after cycles 3 and 6 by radiological imaging tests. Physical examination about target lesion and regional lymph nodes were carried out at every cycle. Breast MRI was used to assess clinical complete response following Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Toxic effects were supervised on an ongoing basis and graded according to Common Terminology Criteria for Adverse Events, version 3.0.

Statistical design
Patients were included in the intent-to treat (ITT) population received all the 6 cycles of apatinib, nabpaclitaxel and carboplatin. Statistical analysis was conducted using SPSS software (version 21.0). The enumeration data are expressed in percentage, and the measurement data are expressed as mean ± standard deviation (SD).

Baseline characteristics
From September 2018 to August 2020, 33 patients were recruited at the First A liated Hospital of Nanjing Medical University and 32 patients completed the planned treatment course that were included in all e cacy and safety analyses. One patient declined further treatment after cycle 4 due to vascular diseases (aortic intramural hematoma) and received Breast conserving surgery, the pathology showed that the invasive cancer tissue was signi cantly reduced, and only a few residual cancer cells were scattered and distributed.
Baseline demographics and disease characteristics of the 32 eligible patients are shown in Table 1. Based on breast MRI, most patients had stage III tumors (65.6%) and the median longest tumor size was 3.5 cm (Table 1).  Table 2 shows the rate of pCR in both breast and axillary lymph nodes was 43.8% (95%CI: 26.4%-62.3%). At the time of surgery, 7 (63.6%) of the 11 patients without axillary lymph node metastasis and 7 (33.3%) patients had axillary lymph node metastasis achieved a pCR (Fig. 1A, Table 2).  (Fig. 1B, Table 2).
Following surgery, besides a pCR rate of 43.8%, most of patients exhibited a ypT stage of T1. E cacy results about tumor shrinkage size are shown in Fig. 2. The median target lesions in breast reduced to 1.2 cm after the third cycle treatment and 0.9cm after the last cycle.
Follow-up is ongoing and the data of disease-free survival (DFS) and overall survival (OS) are not yet published.

Saety
All 32 patients were included in the safety analysis during the treatment period and there were no intolerable AEs after corresponding treatment (Table 3). Non-haematological The chemotherapy dose reduction or delay was calculated (Fig. 3A-C), most commonly because of myelosuppression and abnormal liver function. In total, all the patients received all six cycles of therapy and underwent breast surgery.

Discussion
Although anthracycline and taxane-based neoadjuvant chemotherapy has been the mainstay for TNBC, 30-40% of early stage TNBCs recurred despite receiving the neoadjuvant regimen. Studies such as WSG-ADAPT TN (24) suggest that taxane combines with carboplatinum regimen but neoadjuvant therapy can increase the pCR rate of TNBC. GeparSixto (19) showed that adding platinum to TNBC neoadjuvant therapy can bring longevity bene ts, but the results of the CALGB40603(18) study are the opposite.
Therefore, due to lack of long-term survival and evidence-based medicine reports, whether platinum can be used for neoadjuvant therapy has always been controversial.
Results of our study showed that apatinib combined with albumin paclitaxel and carboplatin as neoadjuvant regimen is a safe and well-tolerated, demonstrating a 43.8% pCR rate in patients with stage II-III TNBC. This data compares favourably with recent published data for neoadjuvant therapy in early TNBC, including a pCR rate of 41% in patients received albumin paclitaxel in the Impassion031 trial (25) and 40.6% in patients with treatment of nab-paclitaxel and carboplatin in NeoTRIP trial. While the increasing pCR rate were observed combining with immunotherapy in the KEYNOTE522(26) and Impassion031 trial (25), it is conceivable that controversy over the addition of immune check point inhibitors would persist due to negative result of the NeoTRIP trial and the urgent need of biomarkers that predict pCR.
Hypothesis of anti-angiogenic therapies play a speci c role in populations of patients with highly proliferative disease. Patients involved in our study were almost locally advanced tumors either with large tumor sizes or a considerable number of lymph node metastases. To the best of our knowledge, no other article has reported data on the combination of neoadjuvant apatinib and chemotherapy exclusively in patients with TNBC. As an oral drug, apatinib in convenient to receive treatment and more economical than other anti-angiogenic therapy. In so doing, we have already explored the curative effect of low dose apatinib (250mg) in patients with advanced HER2-negative breast cancer and proposed patients may bene t more from the therapy, as well as recent research published. Therefore, we attempt to determine whether the addition of apatinib to neoadjuvant chemotherapy produce higher pCR. Previously reported research on the combination of neoadjuvant anti-angiogenic and chemotherapy has shown varied results (20,(27)(28)(29). The difference in terms of pCR rate among the studies could be almost attributed to the patient populations, baseline stage of breast cancer and the combined chemotherapy regimen. The GeparQuinto trial (19) presented a signi cant improvement in pCR rate combining with bevacizumab in TNBC (39% vs 28%, p = 0.003). Meanwhile, the NSABP B-40 trial(20) demonstrated a numerical increase of pCR rate in patients with TNBC without statistical signi cance (52% vs 47%, p = 0.34). Similarly, the KCSG BR-0905(29) shared a comparable pCR rate of 42% in TNBC with those studies. Signi cantly,these data could be attributable to the relatively early-stage patients included in their research and the pCR rate of patients without axillary lymph node metastasis in our research was as high as 63.6%, so caution should be exercised when making comparisons to others.
Another growing attention is apatinib-related postoperative wound complications. Antiangiogenic therapy may increase the risk of postoperative complications, including wound dehiscence and slow recovery, especially patients received breast reconstruction with tissue expanders. A minimum of 21 days between surgery and the last therapy had been recommended to avoid postoperative wound complications in our study and recorded few wound complications with neoadjuvant apatinib indeed. Furthermore, other adverse events reported in our study were all manageable. The most frequently reported grade 3/4 toxicity were thrombopenia, neutropenia, leukopenia and hypertension and a clear upturn was observed following treatment or dose reduction.
The main limitations of our study are the lack of a control arm and the relatively small sample size. A future neoadjuvant study incorporating more information for prospective detection of patients to explore the biomarkers that can simultaneously predict the reaction e cacy and potential toxicity has crucial clinical value.
Apatinib combined with nab-paclitaxel and carboplatin showed an encouraging anti-tumor activity in patients with TNBC resulting in high pCR rate and tolerable side effects toxicity. Our study adds to the evidence supporting the addition of apatinib to neoadjuvant therapy for TNBC, especially locally advanced cancer and provides further rationale for e cient neoadjuvant therapy for patients with relatively severe breast cancer.

Conclusion
Analyses of this phase II study proved that neoadjuvant treatment containing apatinib, nab paclitaxel and carboplatin is e cient in patients of high tumor burden with TNBC, which is a safe and well-tolerated. We demonstrated the pCR rate in patients with stage II-III TNBC is 43.8%and patients without axillary lymph node metastasis among them is as high as 63.6%. We propose that patients with high tumor load may bene t from the neoadjuvant treatment of apatinib combined with nab-paclitaxel and carboplatin. University (2018-SR-115). All human samples and biopsies were collected after obtaining a written informed consent.

Consent for publication
Not Applicable.

Availability of data and materials
The data analysed during the research are included in this study or are available on reasonable request.

Competing interests
The authors declare that there is no competing interests.  Shrinkage size of tumor after neoadjuvant apatinib.