Med Oncol (2015) 32:184 DOI 10.1007/s12032-015-0634-7
Lymphatic-targeted therapy following neoadjuvant chemotherapy: a promising strategy for lymph node-positive breast cancer treatment Jianghao Chen1,4 • Qing Yao1 • Hui Wang1 • Bo Wang2 • Juliang Zhang1 Ting Wang1 • Yonggang Lv1 • Zenghui Han3 • Ling Wang1,4
•
Received: 4 May 2015 / Accepted: 8 May 2015 / Published online: 26 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Neoadjuvant chemotherapy has been increasingly used to downstage breast cancer prior to surgery recently. However, in some cases, it was observed that despite sufficient regression of primary tumors, the metastatic lymph nodes remained nonresponsive. In this study, we applied lymphatic-targeted strategy to evaluate its efficacy and safety for patients presenting refractory nodes following systemic chemotherapy. A total of 318 breast cancer patients were demonstrated with lymph node metastasis by needle biopsy and given neoadjuvant chemotherapy. Two cycles later, 72 patients were observed with responsive tumors but stable nodes, 42 of which received a subcutaneous injection of lymphatic-targeted pegylated liposomal doxorubicin during the third cycle, while the remaining 30 patients were continued with former neoadjuvant therapeutic pattern and regarded as the control. Lymphatic-targeted treatment substantially increased both clinical and pathological node response rate [62 % Jianghao Chen and Qing Yao have contributed equally to this work. & Jianghao Chen [email protected] & Ling Wang [email protected] 1
Department of Thyroid, Breast and Vascular Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi Province, China
2
Department of Epidemiology, The Fourth Military Medical University, Xi’an, Shaanxi Province, China
3
Department of Ultrasound, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi Province, China
4
Department of Vascular and Endocrine Surgery, Xijing Hospital, The Fourth Military Medical University, 17th Changle West Road, Xi’an 710032, Shaanxi Province, China
(26/42) vs. 13 % (4/30) and 12 % (5/42) vs. 0 (0/30), respectively], and induced a higher apoptosis level of metastatic cells (median, 41 vs. 6 %), compared with the control. Moreover, a higher disease-free survival was observed after a median follow-up of 4 years (69 vs. 56 %). Inflammatory reaction surrounding injection sites was the most common side effect. Lymphatic chemotherapy has reliable efficacy and well-tolerated toxicity for breast cancer patients presenting refractory lymph nodes following neoadjuvant chemotherapy. Keywords Breast cancer Neoadjuvant chemotherapy Lymph node metastasis Lymphatic-targeting chemotherapy
Introduction Consistent with common types of carcinoma, breast cancer displays a predilection to metastasize initially into regional lymph nodes [1]. It has been well established that the indirect route via lymph nodes to vital organs contributes obviously to the metastasis of breast cancer. Meanwhile, the involvement ratio of axillary lymph node, always being removed during radical resection, is closely related to the overall survival of node-positive patients [2]. To remove the lymph nodes located in internal mammary, mediastinum, supraclavicle, or hepatic portal, neoadjuvant chemotherapy appears to be a promising approach, better than both modified radical mastectomy and breast-conserving surgery [3]. However, in contrast to favorable response of breast cancer to neoadjuvant chemotherapy, there still remain nonresponsive tumor-involved lymph nodes in some patients, which is likely due to the difficult achievement of drugs by systemic chemotherapy in the
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lymphatic system possessing peculiar nature and anatomy [4]. Hence, seeking for an effective strategy for lymph node-positive breast cancer therapy is of great importance. Early lymph node involvement gives rise to the concept of SLN (sentinel lymph cells), which is the first node in a lymphatic basin receiving lymph flow from primary tumor [5, 6]. The procedure of SLN identification consists of injecting medical dye or radioactive agent subcutaneously (s.c.), into the tissue adjacent to primary tumor, detecting the first group of nodes that are stained or present radioactivity. The method of SLN identification is enabled by the ability of lymphatic capillaries to take up macromolecules and tiny particles from interstitial space through the interstitial-lymphatic fluid transport [7]. SLN identification inspires us that targeting regional lymph node can be attained by interstitial administration of macromolecular or carrier-mediated agents. Previously, we have administered chemotherapeutic drug-loaded liposomes and activated carbon particles, respectively, into breast tumor-bearing animals and patients. Results showed that carboplatin-activated carbon suspension (s.c.) injection of liposomal doxorubicin (LD) of 120 nm in diameter to a metastatic breast cancer model in rabbits notably inhibited proliferation of lymphatically disseminating cells. No evidence of local toxicity, such as allergy, ulceration, or erosion of skin, was found, suggesting that the interstitial injection of LD might be safe [8]. Moreover, in breast cancer patients, compared to carboplatin solution administered intravenously (i.v.), s.c. effectively increased drug retentions in axillary lymph nodes [9]. Nevertheless, in spite of all the exciting findings, the administration of LD to patients via extravascular route is prohibited at present for the likely damage to local tissues. In this study, we applied the lymphatic-targeted therapy, which is proceeded by pegylated liposomal doxorubicin (PLD, Schering Plough, Kenilworth, NJ, USA) s.c., for the breast cancer patients with refractory lymph nodes following preoperative chemotherapy. The efficacy and safety profiles were also assessed to guide the application and feasibility of lymphatic-targeted neoadjuvant chemotherapy in lymph node-positive breast cancer.
Materials and methods Patient population From October 2006 to August 2009, a series of 318 breast cancer patients diagnosed by 11-gauge core needle biopsy were eligible for entry into this study. The lymph node metastasis was detected by fine needle aspiration on the biggest axillary node exhibiting incrassated cortex under ultrasound. Neoadjuvant chemotherapy was applied
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for the first stage of treatment. The inclusion criteria were listed as follows: Eastern Cooperative Oncology Group (ECOG) performance status 0–2, no previous therapy, and adequate heart, hepatic, renal, and hematologic function. This study was approved by the Ethics Committee of Fourth Military Medical University in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. Neoadjuvant treatment strategy Neoadjuvant chemotherapy regimens included DE (docetaxel 75 mg/m2, d1; epirubicin 60 mg/m2, d1, every 3 weeks), CEF (cyclophosphamide 500 mg/m2, d1, 8; epirubicin 60 mg/m2, d1; 5-fluorouracil 600 mg/m2, d1, 8, every 3 weeks), and VP (vinorelbine 25 mg/m2, d1, 8; PLD 35 mg/m2, d1, every 3 weeks). After two cycles of chemotherapy, patients exhibiting NR (no response) in both primary cancer and lymph node were excluded from the study and submitted to other regimens. Patients exhibiting OR (objective response) in both cancer and node were included in this study and separated into two groups. Patients showing responsive cancer but unresponsive node were recommended to receive lymphatic chemotherapy. Lymphatic chemotherapy Lymphatic chemotherapy was performed by an s.c. injection of 4 mg of PLD, which was diluted, immediately before use, with 6 ml of normal saline to adjust the final concentration of doxorubicin to 0.5 mg/ml. The injection site was above the tumor entity. The drug-diffusing region was rubbed gently for 1 min to facilitate drug spread inside subcutaneous lymphatic nets and then covered by towel-packed ice for 2 h to alleviate the local reaction. Treatment was started concurrently with the third chemotherapy cycle and repeated for three times at 1-week intervals. Following the completion of chemotherapy, all patients received a final evaluation and then underwent Patey’s radical mastectomy or lumpectomy plus axillary clearance. For those suitable for breast conservation and having undergone lymphatic chemotherapy, a fusiform incision (not the routinely linear incision) above tumor entity was made to dissect the skin surrounding the injection site. All patients received three cycles of adjuvant chemotherapy. Radiotherapy was given to most (87 %) patients, including all breast conservative cases. Patients with positive estrogen receptor and/or progesterone receptor received 5 years of endocrine therapy. All participants were followed up at 6-month intervals in an outpatient department.
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Efficacy assessment At the time of diagnosis and after each cycle of chemotherapy, the sizes of breast tumors and lymph nodes were measured by physical examination and ultrasound, respectively [10]. The clinical response was classified according to WHO criteria [11]. Cancer or lymph node classified as complete or partial response was defined as OR, and that classified as stable or progressive disease was defined as NR. The evaluation of lymph nodes was only made on those with biopsy-proven metastatic disease. Tissue processing Tissue samples, collected from biopsy or surgery, were fixed in 4 % neutral formalin for 24 h and then embedded in paraffin blocks. Sections were cut at 3 lm and mounted on glass slides overnight at room temperature. Histological classification and grading were made using light microscopy examination of tissue sections stained with H&E. Pathological response of breast tumor was defined as follows: (a) pathological complete response (pCR), no residual viable invasive tumor, i.e., only in situ disease or tumor stroma remained; and (b) nonresponse, any viable tumor in breast. The pCR of lymph nodes was defined as: initially axillary lymph node positive but converted to lymph node Table 1 Baseline patient characteristics
negative after primary therapy [12]. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dTUP nick end labeling (TUNEL) method [8]. Apoptotic index was defined as the ratio of TUNEL-positive cells against total nucleated cells. The status of hormonal receptor and human epidermal growth factor receptor 2 (HER-2) was investigated by immunohistochemistry using a standard avidin–biotin complex technique [13, 14]. Statistical analysis SPSS 11.0 for windows was used for statistical analysis. The significance of differences was determined by one-way ANOVA, Dunnett’s test and Fisher’s exact test. Kaplan– Meier curves and log–rank statistics were used for survival analysis.
Results Clinical results A total of 318 patients were enrolled into this study, following two cycles of primary chemotherapy, 49 patients failed to respond and were excluded from this study. Of the remaining 269 patients, 72 cases showed responsive tumor Lymph-targeted group (%)
Age (years) \35
Control group (%)
4 (9.5)
2 (6.7)
29 (69.0)
22 (73.3)
9 (21.4)
6 (20.0)
Premenopausal
23 (54.8)
15 (50.0)
Perimenopausal
7 (16.7)
7 (23.3)
Postmenopausal
12 (28.6)
8 (26.7)
B2
16 (38.1)
11 (36.7)
[2 and B5
23 (54.8)
17 (56.7)
3 (7.1)
2 (6.7)
35–59 C60 Menopausal status
Tumor size (cm)
[5 Histological grade Grade 1
9 (21.4)
8 (26.7)
Grade 2
24 (57.1)
16 (53.3)
Grade 3
7 (16.7)
6 (20.0)
2 (4.8)
0 (0)
Unknown Hormone status Positive
26 (61.9)
20 (66.7)
Negative
16 (38.1)
10 (33.3)
HER-2 status Positive
8 (19.0)
6 (20.0)
Negative
34 (81.0)
24 (80.0)
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but unresponsive lymph nodes and the baseline characteristics are described in Table 1. Forty-two (35 received DE, 6 CEF, and 1 VP) of them were consented to receive lymphatic chemotherapy; the other 30 (24 received DE, 5 CEF, and 1 VP) were defined as the control group and continued with previous treatment pattern. All the scheme was shown in Fig. 1.
group. Meanwhile, the percentage of lymph node was 9.6 % (19/197) in lymph-targeted group, lower than that of the control group as 11.9 % (5/42, Table 2). No pCR was identified in lymph nodes of control group. Surprisingly, the highest node pCR rate was observed in lymph-targeted group, with significant difference from the control group (p \ 0.0001).
Clinical and pathological response
Apoptosis index
Details of clinical and pathological response are shown in Table 2. The OR rate of breast cancers was 92.9 % (39/42) and 90.0 % (27/30), respectively. The detected lymph nodes were 61.9 % (26/42) and 13.3 % (4/30). A total of 23 tumors and 12 nodes that responded after the second cycle rebounded after the third cycle. No difference was found in tumor OR rate between groups. The node response rate of lymph-targeted group was notably higher than that of control group (p \ 0.0001), in consistent with the results from hybrid 18F-FDG PET/CT imaging before and after lymphatic chemotherapy (Fig. 2).
The apoptotic cells were rarely seen in biopsy tissues, and the median apoptosis index was only reached to 3.2 % (range 1–5 %). However, following neoadjuvant therapy, the median apoptosis index was increased to 16.9 and 18.1 %, respectively, in lymph-targeted group and control group. Besides, the apoptosis occurred in tumor-involved lymph nodes rate was up to 41.3 and 6.1 %, respectively. As indicted, a dramatically higher apoptosis level of lymph nodes was observed in lymph-targeted group (Fig. 3).
Pathological response
Erythema and edema emerged in all cases receiving lymphatic treatment, often of 2–3 cm in diameter, surrounding the injection site, and persisted until the time of surgery. Systemic hematologic and non-hematologic toxicity was
The pCR rate of lymph-targeted group was 7.1 % (3/42), in comparison with the result of 6.7 % (2/30) in control Fig. 1 Research scheme of controlled clinical study
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Adverse events
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Table 2 Overall response to treatment and outcome
Lymph-targeted group (%)
Control group (%)
39 (92.9)
27 (90.0)
3 (7.1)
3 (10.0)
Objective response
26 (61.9)
4 (13.3)
No response
16 (38.1)
26 (86.7)
Conservative
19 (45.2)
13 (43.3)
Mastectomy
23 (54.8)
17 (56.7)
Clinical response of cancer Objective response No response Clinical response of lymph nodes
Surgery
Pathological response of cancer Complete pathological response Residual invasive disease
3 (7.1)
2 (6.7)
39 (92.9)
28 (93.3)
Pathological status of lymph nodes Complete pathological response
5 (11.9)
0 (0)
1–3 4–9
23 (54.8) 10 (23.8)
17 (56.7) 10 (33.3)
C10
4 (9.5)
3 (10.0)
1 year
40 (95.2)
26 (86.7)
2 years
36 (85.7)
21 (70.0)
3 years
32 (75.3)
18 (56.2)
4 years
30 (69.4)
18 (56.2)
1 year
41 (97.6)
29 (96.7)
2 years
37 (88.1)
25 (83.3)
3 years
32 (73.7)
20 (66.1)
4 years
32 (73.7)
20 (66.1)
Progression-free survival
Overall survival
similar in each group (data not shown). One case developed blisters 2 days after the final course of lymphatic chemotherapy and received Patey’s radical mastectomy on the next day, ahead of the scheduled time. The involved skin was contained in the operation field, thereby having little influence on postoperative recovery (Fig. 4b). No evidence of necrosis was found under microscopic examination. Inflammatory reaction, representing as lymphocytes, macrophages, and mast cells gathering at the injection site, was the most frequent finding (Fig. 4a). Survival The median follow-up time was 48 months (range 31–65 months), and there were 80 relapses and 48 deaths. The disease-free survival (DFS) and overall survival (OS) figures are detailed in Fig. 4c, d. The DFS was 69.4 and 56.2 %, respectively, and the OS was 73.7 and 66.1 %, respectively. Compared to control, patients in lymph-targeted group achieved a better DFS (p = 0.036), while no significant improvement was observed in OS (p = 0.387).
Discussion Neoadjuvant chemotherapy has emerged over the past two decades as a new approach for the treatment of breast cancer to eliminate occult systemic diseases, increasing the likelihood of breast conservation, while not affecting the local recurrence rate [15, 16], or compromising survival [17]. However, although tumor shrinkage may be induced in up to 80 % of patients, not all of them achieve isochronous lymph node regression, and the persisting pathological node involvement is always associated with much poor outcome [18]. Presently, SLN identification inspires us that targeting regional lymph node can be attained by interstitial administration of macromolecular or carrier-mediated agents and liposomes was selected as an ideal carrier. Although their extravascular administration is by far prohibited, what we observed in this study supported our previous judgment that the risk is controllable. Except for local blisters in one case, the most common complication was inflammatory reaction and no evidence of necrosis was detected histopathologically. All adverse
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Fig. 2 Hybrid 18F-FDG PET/CT imaging before and after lymphatic chemotherapy. PET imaging demonstrated an evident radioactivity of 18FFDG in supraclavicular lymph node (a). After lymphatic chemotherapy, FDG radioactivity disappeared (b)
Fig. 3 Apoptosis in breast cancer and lymph node. a The TUNELpositive cells in the primary tumor specimens. b The apoptotic cells in axillary lymph nodes after lymphatic-targeted therapy. c The
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percentage of apoptotic cells before and after the different neoadjuvant therapy regimen, HE 9 200
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Fig. 4 Adverse events of lymphatic chemotherapy and Kaplan–Meier estimates of disease-free survival and overall survival. a Erythema emerged in all cases after receiving the subcutaneous injection of pegylated liposomal doxorubicin. b Inflammatory reaction, representing as lymphocytes (green arrow), macrophages (yellow arrow), and mast cells (red arrow) gathering at the injection site, was the most frequent finding under microscopic examination, HE 9 100. c Disease-free survival for groups with a median follow-up of 4 years. d Overall survival for groups with a median follow-up of 4 years
events were limited in drug-diffusing region and were handled satisfactorily by removing the involved skin. A challenge probably encountered during lymphatic chemotherapy is the occlusion of lymph vessels by emboli of metastatic cells, which will block the spread of agents into infected nodes. Under such circumstance, we insist that our approach still has its strength. First of all, the tumor-associated lymphangiogenesis, usually at the periphery of tumor, provides alternative paths for drug ferry from tumor site to lymph nodes. Secondly, a part of liposomes can be phagocytized by inflammatory cells such as macrophages and dendritic cells and then transferred to neighboring lymph nodes [19–22]. Moreover, the continuous LD accumulation will gradually clean up cancer cells occluded in lymphatic vessels. Finally, the most important one, the skin surrounding the injection site will be dissected in surgery, reducing the possible hazard to an acceptable level. In addition to all the advantages mentioned above, lymphatic treatment may furnish additional benefits for patients receiving breast conservative surgery. We speculate that it also plays a role in cancer recurrence,
especially for cancers in non-outer upper quadrant. In these cases, lymphatic chemotherapy provides a chance to clean up lymphatic vessels prior to surgery. The liposomal encapsulation provides a longer duration of drug exposure to tumor tissue. Furthermore, liposomal could selectively accumulate in regional lymphatic tissues, resulting in a notably higher drug concentration than systemic administration. The in vitro and clinical studies by Zamboni et al. revealed that liposomal formulation might to some extent overcome tumor resistance to conventional doxorubicin [23, 24]. Taken together, we explored the feasibility of applying lymphatic-targeted therapy via interstitial injection of PLD for breast cancer patients who demonstrated refractory lymph nodes following preoperative chemotherapy. Our data supported that lymphatic chemotherapy can be used in neoadjuvant setting as a safe and effective component of the approach to the systemic therapy to sterilize lymph node metastases and achieve better disease-free survival. However, we did not assess systemic toxicities in this study, since the dose of PLD s.c. was very limited. The details of performing lymphatic therapy, including the dose
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and concentration of PLD, the interval, and number of cycles of treatment, were based on our preliminary experience. The present results merit further study to optimize lymphatic therapy procedures. Acknowledgments This work was supported by Grants provided by National Natural Science Foundation of China (No. 81172510, JC; No. 81272899, LW) and Natural Science Foundation of Shaanxi Province (No. 2011K12-45, JC; No. 2012K13-02-28, QY). Conflict of interest of interest.
The authors declare that they have no conflict
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Lymphatic-targeted therapy following neoadjuvant chemotherapy: a promising strategy for lymph node-positive breast cancer treatment Jianghao Chen1,4 • Qing Yao1 • Hui Wang1 • Bo Wang2 • Juliang Zhang1 Ting Wang1 • Yonggang Lv1 • Zenghui Han3 • Ling Wang1,4
•
Received: 4 May 2015 / Accepted: 8 May 2015 / Published online: 26 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Neoadjuvant chemotherapy has been increasingly used to downstage breast cancer prior to surgery recently. However, in some cases, it was observed that despite sufficient regression of primary tumors, the metastatic lymph nodes remained nonresponsive. In this study, we applied lymphatic-targeted strategy to evaluate its efficacy and safety for patients presenting refractory nodes following systemic chemotherapy. A total of 318 breast cancer patients were demonstrated with lymph node metastasis by needle biopsy and given neoadjuvant chemotherapy. Two cycles later, 72 patients were observed with responsive tumors but stable nodes, 42 of which received a subcutaneous injection of lymphatic-targeted pegylated liposomal doxorubicin during the third cycle, while the remaining 30 patients were continued with former neoadjuvant therapeutic pattern and regarded as the control. Lymphatic-targeted treatment substantially increased both clinical and pathological node response rate [62 % Jianghao Chen and Qing Yao have contributed equally to this work. & Jianghao Chen [email protected] & Ling Wang [email protected] 1
Department of Thyroid, Breast and Vascular Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi Province, China
2
Department of Epidemiology, The Fourth Military Medical University, Xi’an, Shaanxi Province, China
3
Department of Ultrasound, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi Province, China
4
Department of Vascular and Endocrine Surgery, Xijing Hospital, The Fourth Military Medical University, 17th Changle West Road, Xi’an 710032, Shaanxi Province, China
(26/42) vs. 13 % (4/30) and 12 % (5/42) vs. 0 (0/30), respectively], and induced a higher apoptosis level of metastatic cells (median, 41 vs. 6 %), compared with the control. Moreover, a higher disease-free survival was observed after a median follow-up of 4 years (69 vs. 56 %). Inflammatory reaction surrounding injection sites was the most common side effect. Lymphatic chemotherapy has reliable efficacy and well-tolerated toxicity for breast cancer patients presenting refractory lymph nodes following neoadjuvant chemotherapy. Keywords Breast cancer Neoadjuvant chemotherapy Lymph node metastasis Lymphatic-targeting chemotherapy
Introduction Consistent with common types of carcinoma, breast cancer displays a predilection to metastasize initially into regional lymph nodes [1]. It has been well established that the indirect route via lymph nodes to vital organs contributes obviously to the metastasis of breast cancer. Meanwhile, the involvement ratio of axillary lymph node, always being removed during radical resection, is closely related to the overall survival of node-positive patients [2]. To remove the lymph nodes located in internal mammary, mediastinum, supraclavicle, or hepatic portal, neoadjuvant chemotherapy appears to be a promising approach, better than both modified radical mastectomy and breast-conserving surgery [3]. However, in contrast to favorable response of breast cancer to neoadjuvant chemotherapy, there still remain nonresponsive tumor-involved lymph nodes in some patients, which is likely due to the difficult achievement of drugs by systemic chemotherapy in the
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lymphatic system possessing peculiar nature and anatomy [4]. Hence, seeking for an effective strategy for lymph node-positive breast cancer therapy is of great importance. Early lymph node involvement gives rise to the concept of SLN (sentinel lymph cells), which is the first node in a lymphatic basin receiving lymph flow from primary tumor [5, 6]. The procedure of SLN identification consists of injecting medical dye or radioactive agent subcutaneously (s.c.), into the tissue adjacent to primary tumor, detecting the first group of nodes that are stained or present radioactivity. The method of SLN identification is enabled by the ability of lymphatic capillaries to take up macromolecules and tiny particles from interstitial space through the interstitial-lymphatic fluid transport [7]. SLN identification inspires us that targeting regional lymph node can be attained by interstitial administration of macromolecular or carrier-mediated agents. Previously, we have administered chemotherapeutic drug-loaded liposomes and activated carbon particles, respectively, into breast tumor-bearing animals and patients. Results showed that carboplatin-activated carbon suspension (s.c.) injection of liposomal doxorubicin (LD) of 120 nm in diameter to a metastatic breast cancer model in rabbits notably inhibited proliferation of lymphatically disseminating cells. No evidence of local toxicity, such as allergy, ulceration, or erosion of skin, was found, suggesting that the interstitial injection of LD might be safe [8]. Moreover, in breast cancer patients, compared to carboplatin solution administered intravenously (i.v.), s.c. effectively increased drug retentions in axillary lymph nodes [9]. Nevertheless, in spite of all the exciting findings, the administration of LD to patients via extravascular route is prohibited at present for the likely damage to local tissues. In this study, we applied the lymphatic-targeted therapy, which is proceeded by pegylated liposomal doxorubicin (PLD, Schering Plough, Kenilworth, NJ, USA) s.c., for the breast cancer patients with refractory lymph nodes following preoperative chemotherapy. The efficacy and safety profiles were also assessed to guide the application and feasibility of lymphatic-targeted neoadjuvant chemotherapy in lymph node-positive breast cancer.
Materials and methods Patient population From October 2006 to August 2009, a series of 318 breast cancer patients diagnosed by 11-gauge core needle biopsy were eligible for entry into this study. The lymph node metastasis was detected by fine needle aspiration on the biggest axillary node exhibiting incrassated cortex under ultrasound. Neoadjuvant chemotherapy was applied
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for the first stage of treatment. The inclusion criteria were listed as follows: Eastern Cooperative Oncology Group (ECOG) performance status 0–2, no previous therapy, and adequate heart, hepatic, renal, and hematologic function. This study was approved by the Ethics Committee of Fourth Military Medical University in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. Neoadjuvant treatment strategy Neoadjuvant chemotherapy regimens included DE (docetaxel 75 mg/m2, d1; epirubicin 60 mg/m2, d1, every 3 weeks), CEF (cyclophosphamide 500 mg/m2, d1, 8; epirubicin 60 mg/m2, d1; 5-fluorouracil 600 mg/m2, d1, 8, every 3 weeks), and VP (vinorelbine 25 mg/m2, d1, 8; PLD 35 mg/m2, d1, every 3 weeks). After two cycles of chemotherapy, patients exhibiting NR (no response) in both primary cancer and lymph node were excluded from the study and submitted to other regimens. Patients exhibiting OR (objective response) in both cancer and node were included in this study and separated into two groups. Patients showing responsive cancer but unresponsive node were recommended to receive lymphatic chemotherapy. Lymphatic chemotherapy Lymphatic chemotherapy was performed by an s.c. injection of 4 mg of PLD, which was diluted, immediately before use, with 6 ml of normal saline to adjust the final concentration of doxorubicin to 0.5 mg/ml. The injection site was above the tumor entity. The drug-diffusing region was rubbed gently for 1 min to facilitate drug spread inside subcutaneous lymphatic nets and then covered by towel-packed ice for 2 h to alleviate the local reaction. Treatment was started concurrently with the third chemotherapy cycle and repeated for three times at 1-week intervals. Following the completion of chemotherapy, all patients received a final evaluation and then underwent Patey’s radical mastectomy or lumpectomy plus axillary clearance. For those suitable for breast conservation and having undergone lymphatic chemotherapy, a fusiform incision (not the routinely linear incision) above tumor entity was made to dissect the skin surrounding the injection site. All patients received three cycles of adjuvant chemotherapy. Radiotherapy was given to most (87 %) patients, including all breast conservative cases. Patients with positive estrogen receptor and/or progesterone receptor received 5 years of endocrine therapy. All participants were followed up at 6-month intervals in an outpatient department.
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Efficacy assessment At the time of diagnosis and after each cycle of chemotherapy, the sizes of breast tumors and lymph nodes were measured by physical examination and ultrasound, respectively [10]. The clinical response was classified according to WHO criteria [11]. Cancer or lymph node classified as complete or partial response was defined as OR, and that classified as stable or progressive disease was defined as NR. The evaluation of lymph nodes was only made on those with biopsy-proven metastatic disease. Tissue processing Tissue samples, collected from biopsy or surgery, were fixed in 4 % neutral formalin for 24 h and then embedded in paraffin blocks. Sections were cut at 3 lm and mounted on glass slides overnight at room temperature. Histological classification and grading were made using light microscopy examination of tissue sections stained with H&E. Pathological response of breast tumor was defined as follows: (a) pathological complete response (pCR), no residual viable invasive tumor, i.e., only in situ disease or tumor stroma remained; and (b) nonresponse, any viable tumor in breast. The pCR of lymph nodes was defined as: initially axillary lymph node positive but converted to lymph node Table 1 Baseline patient characteristics
negative after primary therapy [12]. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dTUP nick end labeling (TUNEL) method [8]. Apoptotic index was defined as the ratio of TUNEL-positive cells against total nucleated cells. The status of hormonal receptor and human epidermal growth factor receptor 2 (HER-2) was investigated by immunohistochemistry using a standard avidin–biotin complex technique [13, 14]. Statistical analysis SPSS 11.0 for windows was used for statistical analysis. The significance of differences was determined by one-way ANOVA, Dunnett’s test and Fisher’s exact test. Kaplan– Meier curves and log–rank statistics were used for survival analysis.
Results Clinical results A total of 318 patients were enrolled into this study, following two cycles of primary chemotherapy, 49 patients failed to respond and were excluded from this study. Of the remaining 269 patients, 72 cases showed responsive tumor Lymph-targeted group (%)
Age (years) \35
Control group (%)
4 (9.5)
2 (6.7)
29 (69.0)
22 (73.3)
9 (21.4)
6 (20.0)
Premenopausal
23 (54.8)
15 (50.0)
Perimenopausal
7 (16.7)
7 (23.3)
Postmenopausal
12 (28.6)
8 (26.7)
B2
16 (38.1)
11 (36.7)
[2 and B5
23 (54.8)
17 (56.7)
3 (7.1)
2 (6.7)
35–59 C60 Menopausal status
Tumor size (cm)
[5 Histological grade Grade 1
9 (21.4)
8 (26.7)
Grade 2
24 (57.1)
16 (53.3)
Grade 3
7 (16.7)
6 (20.0)
2 (4.8)
0 (0)
Unknown Hormone status Positive
26 (61.9)
20 (66.7)
Negative
16 (38.1)
10 (33.3)
HER-2 status Positive
8 (19.0)
6 (20.0)
Negative
34 (81.0)
24 (80.0)
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but unresponsive lymph nodes and the baseline characteristics are described in Table 1. Forty-two (35 received DE, 6 CEF, and 1 VP) of them were consented to receive lymphatic chemotherapy; the other 30 (24 received DE, 5 CEF, and 1 VP) were defined as the control group and continued with previous treatment pattern. All the scheme was shown in Fig. 1.
group. Meanwhile, the percentage of lymph node was 9.6 % (19/197) in lymph-targeted group, lower than that of the control group as 11.9 % (5/42, Table 2). No pCR was identified in lymph nodes of control group. Surprisingly, the highest node pCR rate was observed in lymph-targeted group, with significant difference from the control group (p \ 0.0001).
Clinical and pathological response
Apoptosis index
Details of clinical and pathological response are shown in Table 2. The OR rate of breast cancers was 92.9 % (39/42) and 90.0 % (27/30), respectively. The detected lymph nodes were 61.9 % (26/42) and 13.3 % (4/30). A total of 23 tumors and 12 nodes that responded after the second cycle rebounded after the third cycle. No difference was found in tumor OR rate between groups. The node response rate of lymph-targeted group was notably higher than that of control group (p \ 0.0001), in consistent with the results from hybrid 18F-FDG PET/CT imaging before and after lymphatic chemotherapy (Fig. 2).
The apoptotic cells were rarely seen in biopsy tissues, and the median apoptosis index was only reached to 3.2 % (range 1–5 %). However, following neoadjuvant therapy, the median apoptosis index was increased to 16.9 and 18.1 %, respectively, in lymph-targeted group and control group. Besides, the apoptosis occurred in tumor-involved lymph nodes rate was up to 41.3 and 6.1 %, respectively. As indicted, a dramatically higher apoptosis level of lymph nodes was observed in lymph-targeted group (Fig. 3).
Pathological response
Erythema and edema emerged in all cases receiving lymphatic treatment, often of 2–3 cm in diameter, surrounding the injection site, and persisted until the time of surgery. Systemic hematologic and non-hematologic toxicity was
The pCR rate of lymph-targeted group was 7.1 % (3/42), in comparison with the result of 6.7 % (2/30) in control Fig. 1 Research scheme of controlled clinical study
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Adverse events
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Table 2 Overall response to treatment and outcome
Lymph-targeted group (%)
Control group (%)
39 (92.9)
27 (90.0)
3 (7.1)
3 (10.0)
Objective response
26 (61.9)
4 (13.3)
No response
16 (38.1)
26 (86.7)
Conservative
19 (45.2)
13 (43.3)
Mastectomy
23 (54.8)
17 (56.7)
Clinical response of cancer Objective response No response Clinical response of lymph nodes
Surgery
Pathological response of cancer Complete pathological response Residual invasive disease
3 (7.1)
2 (6.7)
39 (92.9)
28 (93.3)
Pathological status of lymph nodes Complete pathological response
5 (11.9)
0 (0)
1–3 4–9
23 (54.8) 10 (23.8)
17 (56.7) 10 (33.3)
C10
4 (9.5)
3 (10.0)
1 year
40 (95.2)
26 (86.7)
2 years
36 (85.7)
21 (70.0)
3 years
32 (75.3)
18 (56.2)
4 years
30 (69.4)
18 (56.2)
1 year
41 (97.6)
29 (96.7)
2 years
37 (88.1)
25 (83.3)
3 years
32 (73.7)
20 (66.1)
4 years
32 (73.7)
20 (66.1)
Progression-free survival
Overall survival
similar in each group (data not shown). One case developed blisters 2 days after the final course of lymphatic chemotherapy and received Patey’s radical mastectomy on the next day, ahead of the scheduled time. The involved skin was contained in the operation field, thereby having little influence on postoperative recovery (Fig. 4b). No evidence of necrosis was found under microscopic examination. Inflammatory reaction, representing as lymphocytes, macrophages, and mast cells gathering at the injection site, was the most frequent finding (Fig. 4a). Survival The median follow-up time was 48 months (range 31–65 months), and there were 80 relapses and 48 deaths. The disease-free survival (DFS) and overall survival (OS) figures are detailed in Fig. 4c, d. The DFS was 69.4 and 56.2 %, respectively, and the OS was 73.7 and 66.1 %, respectively. Compared to control, patients in lymph-targeted group achieved a better DFS (p = 0.036), while no significant improvement was observed in OS (p = 0.387).
Discussion Neoadjuvant chemotherapy has emerged over the past two decades as a new approach for the treatment of breast cancer to eliminate occult systemic diseases, increasing the likelihood of breast conservation, while not affecting the local recurrence rate [15, 16], or compromising survival [17]. However, although tumor shrinkage may be induced in up to 80 % of patients, not all of them achieve isochronous lymph node regression, and the persisting pathological node involvement is always associated with much poor outcome [18]. Presently, SLN identification inspires us that targeting regional lymph node can be attained by interstitial administration of macromolecular or carrier-mediated agents and liposomes was selected as an ideal carrier. Although their extravascular administration is by far prohibited, what we observed in this study supported our previous judgment that the risk is controllable. Except for local blisters in one case, the most common complication was inflammatory reaction and no evidence of necrosis was detected histopathologically. All adverse
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Fig. 2 Hybrid 18F-FDG PET/CT imaging before and after lymphatic chemotherapy. PET imaging demonstrated an evident radioactivity of 18FFDG in supraclavicular lymph node (a). After lymphatic chemotherapy, FDG radioactivity disappeared (b)
Fig. 3 Apoptosis in breast cancer and lymph node. a The TUNELpositive cells in the primary tumor specimens. b The apoptotic cells in axillary lymph nodes after lymphatic-targeted therapy. c The
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percentage of apoptotic cells before and after the different neoadjuvant therapy regimen, HE 9 200
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Fig. 4 Adverse events of lymphatic chemotherapy and Kaplan–Meier estimates of disease-free survival and overall survival. a Erythema emerged in all cases after receiving the subcutaneous injection of pegylated liposomal doxorubicin. b Inflammatory reaction, representing as lymphocytes (green arrow), macrophages (yellow arrow), and mast cells (red arrow) gathering at the injection site, was the most frequent finding under microscopic examination, HE 9 100. c Disease-free survival for groups with a median follow-up of 4 years. d Overall survival for groups with a median follow-up of 4 years
events were limited in drug-diffusing region and were handled satisfactorily by removing the involved skin. A challenge probably encountered during lymphatic chemotherapy is the occlusion of lymph vessels by emboli of metastatic cells, which will block the spread of agents into infected nodes. Under such circumstance, we insist that our approach still has its strength. First of all, the tumor-associated lymphangiogenesis, usually at the periphery of tumor, provides alternative paths for drug ferry from tumor site to lymph nodes. Secondly, a part of liposomes can be phagocytized by inflammatory cells such as macrophages and dendritic cells and then transferred to neighboring lymph nodes [19–22]. Moreover, the continuous LD accumulation will gradually clean up cancer cells occluded in lymphatic vessels. Finally, the most important one, the skin surrounding the injection site will be dissected in surgery, reducing the possible hazard to an acceptable level. In addition to all the advantages mentioned above, lymphatic treatment may furnish additional benefits for patients receiving breast conservative surgery. We speculate that it also plays a role in cancer recurrence,
especially for cancers in non-outer upper quadrant. In these cases, lymphatic chemotherapy provides a chance to clean up lymphatic vessels prior to surgery. The liposomal encapsulation provides a longer duration of drug exposure to tumor tissue. Furthermore, liposomal could selectively accumulate in regional lymphatic tissues, resulting in a notably higher drug concentration than systemic administration. The in vitro and clinical studies by Zamboni et al. revealed that liposomal formulation might to some extent overcome tumor resistance to conventional doxorubicin [23, 24]. Taken together, we explored the feasibility of applying lymphatic-targeted therapy via interstitial injection of PLD for breast cancer patients who demonstrated refractory lymph nodes following preoperative chemotherapy. Our data supported that lymphatic chemotherapy can be used in neoadjuvant setting as a safe and effective component of the approach to the systemic therapy to sterilize lymph node metastases and achieve better disease-free survival. However, we did not assess systemic toxicities in this study, since the dose of PLD s.c. was very limited. The details of performing lymphatic therapy, including the dose
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and concentration of PLD, the interval, and number of cycles of treatment, were based on our preliminary experience. The present results merit further study to optimize lymphatic therapy procedures. Acknowledgments This work was supported by Grants provided by National Natural Science Foundation of China (No. 81172510, JC; No. 81272899, LW) and Natural Science Foundation of Shaanxi Province (No. 2011K12-45, JC; No. 2012K13-02-28, QY). Conflict of interest of interest.
The authors declare that they have no conflict
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