Deep learning-assisted monitoring of trastuzumab efficacy in HER2-Overexpressing breast cancer via SERS immunoassays of tumor-derived urinary exosomal biomarkers.

Monitoring drug efficacy is significant in the current concept of companion diagnostics in metastatic breast cancer. Trastuzumab, a drug targeting human epidermal growth factor receptor 2 (HER2), is an effective treatment for metastatic breast cancer. However, some patients develop resistance to this therapy; therefore, monitoring its efficacy is essential. Here, we describe a deep learning-assisted monitoring of trastuzumab efficacy based on a surface-enhanced Raman spectroscopy (SERS) immunoassay against HER2-overexpressing mouse urinary exosomes. Individual Raman reporters bearing the desired SERS tag and exosome capture substrate were prepared for the SERS immunoassay; SERS tag signals were collected to prepare deep learning training data. Using this deep learning algorithm, various complicated mixtures of SERS tags were successfully quantified and classified. Exosomal antigen levels of five types of cell-derived exosomes were determined using SERS-deep learning analysis and compared with those obtained via quantitative reverse transcription polymerase chain reaction and western blot analysis. Finally, drug efficacy was monitored via SERS-deep learning analysis using urinary exosomes from trastuzumab-treated mice. Use of this monitoring system should allow proactive responses to any treatment-resistant issues.

Dual-Targeted Therapy in HER2-Overexpressing Breast Cancer with Trastuzumab and Novel Cholesterol-Based Nioplexes Silencing Mcl-1.

The challenge in HER2-overexpressing breast cancer therapy lies in creating an effective target therapy to overcome treatment resistance. Monoclonal antibodies and target gene silencing by siRNA are two potential strategies that have been widely developed for treating HER2-positive breast cancer. The siRNA delivery system is a crucial factor that influences siRNA therapy's success. In this study, lipid-based nanoparticles (cationic niosomes) composed of different cholesterol-based cationic lipids were formulated and characterized for delivering siRNA into HER2-overexpressing breast cancer cells. Niosomes containing a trimethylammonium headgroup showed the highest siRNA delivery efficiency with low toxicity. The myeloid cell leukemia-1 (Mcl-1) siRNA nioplex treatment significantly decreased mRNA expression and breast cancer cell growth. Dual-targeted therapy, consisting of treatment with an Mcl-1 siRNA nioplex and trastuzumab (TZ) solution, noticeably promoted cell-growth inhibition and apoptosis. The synergistic effect of dual therapy was also demonstrated by computer modeling software (CompuSyn version 1.0). These findings suggest that the developed cationic niosomes were effective nanocarriers for siRNA delivery in breast cancer cells. Furthermore, the Mcl-1 nioplex/TZ dual treatment establishes a synergistic outcome that may have the potential to treat HER2-overexpressing breast cancer.

Inhibiting HER3 Hyperphosphorylation in HER2-Overexpressing Breast Cancer through Multimodal Therapy with Branched Gold Nanoshells.

Treatment failure in breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) is associated mainly to the upregulation of human epidermal growth factor receptor 3 (HER3) oncoprotein linked to chemoresitence. Therefore, to increase patient survival, here a multimodal theranostic nanoplatform targeting both HER2 and HER3 is developed. This consists of doxorubicin-loaded branched gold nanoshells functionalized with the near-infrared (NIR) fluorescent dye indocyanine green, a small interfering RNA (siRNA) against HER3, and the HER2-specific antibody Transtuzumab, able to provide a combined therapeutic outcome (chemo- and photothermal activities, RNA silencing, and immune response). In vitro assays in HER2+ /HER3+ SKBR-3 breast cancer cells have shown an effective silencing of HER3 by the released siRNA and an inhibition of HER2 oncoproteins provided by Trastuzumab, along with a decrease of the serine/threonine protein kinase Akt (p-AKT) typically associated with cell survival and proliferation, which helps to overcome doxorubicin chemoresistance. Conversely, adding the NIR light therapy, an increment in p-AKT concentration is observed, although HER2/HER3 inhibitions are maintained for 72 h. Finally, in vivo studies in a tumor-bearing mice model display a significant progressively decrease of the tumor volume after nanoparticle administration and subsequent NIR light irradiation, confirming the potential efficacy of the hybrid nanocarrier.

Adjuvant treatment with paclitaxel plus trastuzumab for node-negative breast cancer: real-life experience.

Background: In node-negative HER2-overexpressed breast cancers, adjuvant paclitaxel plus trastuzumab treatment is a successful de-escalation approach with excellent survival outcomes. Methods: All patients with HER2+ breast cancer treated in our centers were retrospectively reviewed. Results: We analyzed 173 patients who were treated with adjuvant paclitaxel plus trastuzumab. The mean tumor size was 2.2 cm. There were eight invasive disease events or death: four distant recurrences (2.3%), three locoregional recurrences (1.7%) and one death without documented recurrence after a 52 month follow-up. The 3-year disease-free survival and recurrence-free interval rate was 96.6%. Conclusion: This real-life experience with adjuvant paclitaxel plus trastuzumab demonstrated few distant recurrences and is compatible with the APT trial findings.

Lay abstract In oncology practice, there have been some efforts to avoid the toxicity of combination chemotherapies and reduce the amount of treatment given in recent decades. These strategies have been studied especially for patients with a specific subtype of early-stage breast cancer. We present the results from patients treated in our centers and discuss them in relation to the literature.

Antihypertensive medications and risks of recurrence and mortality in luminal, triple-negative, and HER2-overexpressing breast cancer.

PURPOSE: Antihypertensives are commonly prescribed medications and their effect on breast cancer recurrence and mortality is not clear, particularly among specific molecular subtypes of breast cancer: luminal, triple-negative (TN), and HER2-overexpressing (H2E). METHODS: A population-based prospective cohort study of women aged 20-69 diagnosed with a first primary invasive breast cancer between 2004 and 2015 was conducted in the Seattle, Washington and Albuquerque, New Mexico greater metropolitan areas. Multivariable-adjusted Cox proportional hazards regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for risks of breast cancer recurrence, breast cancer-specific mortality, and all-cause mortality associated with hypertension and antihypertensives. RESULTS: In this sample of 2,383 luminal, 1,559 TN, and 615 H2E breast cancer patients, overall median age was 52 (interquartile range, 44-60). Hypertension and current use of antihypertensives were associated with increased risks of all-cause mortality in each subtype. Current use of angiotensin-converting enzyme inhibitors was associated with increased risks of both recurrence and breast cancer-specific mortality among luminal patients (HR: 2.5; 95% CI: 1.5, 4.3 and HR: 1.9; 95% CI: 1.2, 3.0, respectively). Among H2E patients, current use of calcium channel blockers was associated with an increased risk of breast cancer-specific mortality (HR: 1.8; 95% CI: 0.6, 5.4). CONCLUSION: Our findings suggest that some antihypertensive medications may be associated with adverse breast cancer outcomes among women with certain molecular subtypes. Additional studies are needed to confirm these findings.

Evaluation of the antimetastatic and anticancer activities of morin in HER2­overexpressing breast cancer SK­BR­3 cells.

Morin (2',3,4',5,7­pentahydroxyflavone), a flavonoid isolated from members of the Moraceae family and the leaves of Cudranaia tricuspidata Buread, is a well­known natural substance with anti­inflammatory, antioxidative, antimetastasis, and anticancer effects. However, its anticancer activity has not been comprehensively investigated in human epidermal growth factor receptor 2 (HER2)­overexpressing breast cancer cells. Here, we evaluated the effects of morin on metastasis and cell viability in HER­2­overexpressing human breast cancer SK­BR­3 cells. Our results revealed that morin (150­200 µM) prevented endothelial growth factor (EGF)­induced metastatic potential and suppressed cell migration and MMP­9 activity by inhibiting the EGFR signaling pathway in SK­BR­3 cells by gelatin zymography, wound healing assay and western blotting. Interestingly, morin­induced reductions in cell viability were found to be associated with inhibition of the HER2/EGFR signaling pathway by sulforhodamine B assay and western blotting. Morin also induced the phosphorylation of H2A.X and downregulated the expression levels of RAD51 and survivin, which implied morin­induced DNA damage and that this damage accumulated in HER­2­overexpressing SK­BR­3 cells. Western blot analysis and fluorescent immunocytochemisty showed that morin also activated autophagy after 24 h of treatment and this was maintained at 48 h when activation of apoptosis via PARP cleavage resulted in the activation of caspase­3 and ­7, which was associated with the release of cytochrome c to the cytosol from mitochondria. In addition, the phosphorylation of p38 and JNK was enhanced in the HER­2­overexpressing SK­BR­3 cells by morin after 24 and 48 h of treatment, which suggested p38 and JNK participate in morin­induced cell death. Taken together, the present investigation indicates that morin is a powerful therapeutic candidate for the treatment of HER2­overexpressing breast cancer because it suppresses the EGFR signaling pathway, induces cell death by inhibiting the HER2/EGFR signaling pathway, and suppresses metastatic potential.

Arctigenin-mediated cell death of SK-BR-3 cells is caused by HER2 inhibition and autophagy-linked apoptosis.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is well-known as the therapeutic marker in breast cancer. Therefore, we evaluated anti-cancer activity of arctigenin (ATG) on in SK-BR-3 HER2-overexpressing human breast cancer cells. METHODS: Cell viability and cytotoxicity were analyzed with MTT and colony-forming assay and cell cycle analysis was performed by flow cytometry. The expression and/or phosphorylation of proteins in whole cell lysate and mitochondrial fraction were analyzed by Western blotting. Cellular levels of LC3 and sequestosome 1 (SQSTM1/P62) were observed by immunofluorescence analysis. RESULTS: The result showed that ATG decreased cell viability of SK-BR-3 cells in a concentration-dependent manner. Moreover, ATG increased the sub G1 population linked to the suppression of HER2/EGFR1 signaling pathway. Furthermore, ATG increased the phosphorylation of H2AX and down-regulated RAD51 and survivin expressions, indicating that ATG induced DNA damage and inhibited the DNA repair system. We also found that cleavages of caspase-7 and PARP by releasing mitochondrial cytochrome c into the cytoplasm were induced by ATG treatment for 72 h through the reduction of Bcl-2 and Bcl-xL levels in mitochondria. In contrast, the levels of LC-3 and SQSTM1/P62 were increased by ATG for 24 h through the Akt/mTOR and AMPK signaling pathway. CONCLUSIONS: Taken together, this study indicates that autophagy-linked apoptosis is responsible for the anti-cancer activity of ATG in SK-BR-3 cells, and suggests that ATG is considered a potential therapeutic for the treatment of HER2-overexpressing breast cancer.

Pathological complete response rates with pertuzumab-based neoadjuvant chemotherapy in breast cancer: A single-center experience.

BACKGROUND: Pertuzumab-based neoadjuvant chemotherapy (NAC) has demonstrated successful pathologic complete response (pCR) rates when administered to patients with human epidermal growth factor receptor 2 (HER2)-positive, locally advanced breast cancer and has become standard of care. This study aimed to identify pCR rates in patients receiving a variety of pertuzumab-based NAC regimens. The effect of the addition of an anthracycline and impact of anthracycline and taxane sequencing on pCR was also assessed. METHODS: A retrospective, single-center review was conducted on patients with operable, human epidermal growth factor receptor 2 (HER2)-positive breast cancer that received one of five pertuzumab-containing NAC regimens followed by definitive surgery. RESULTS: Ninety-six patients were included in the analysis; overall, pCR was attained in 49 patients (51%). Of the 61 patients who received an anthracycline-containing NAC regimen, 30 (49%) attained a pCR. Of the 35 patients who received the non-anthracycline NAC regimen, 19 (54%) attained a pCR; difference in pCR was not statistically significant (p = 0.63). Anthracycline/taxane sequence analysis showed that of the patients attaining pCR with an anthracycline-containing NAC, 77% of patients received the taxane portion upfront (p = 0.17). Relative dose intensity of the anthracycline portion was similar irrespective of treatment sequence. However, relative dose intensity of the taxane portion was decreased with upfront anthracycline administration. CONCLUSION: These findings support current recommendations of adding pertuzumab to established regimens for treatment of locally advanced, HER2-positive, early stage breast cancer. The benefit of adding an anthracycline in the neoadjuvant setting remains unclear. Patients treated with the taxane portion of NAC upfront appeared to have a higher rate of pCR and better relative dose intensity than patients who received the anthracycline portion upfront, but differences were not statistically significant. These findings should be verified in a prospective clinical trial.

Pulsed-laser irradiation of multifunctional gold nanoshells to overcome trastuzumab resistance in HER2-overexpressing breast cancer.

BACKGROUND: HER2-overexpressing metastatic breast cancers are challenging practice in oncology when they become resistant to anti-HER2 therapies such as trastuzumab. In these clinical situations, HER2-overexpression persists in metastatic localizations, and can thus be used for active targeting using innovative therapeutic approaches. Functionalized gold nanoparticles with anti-HER2 antibody can be stimulated by near-infrared light to induce hyperthermia. METHODS: Here, hybrid anti-HER2 gold nanoshells were engineered for photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer xenografts. RESULTS: When gold nanoshells were administered in HER2-tumor xenografts, no toxicity was observed. A detailed pharmacokinetic study showed a time-dependent accumulation of gold nanoshells within the tumors, significantly greater with functionalized gold nanoshells at 72 h. This enabled us to optimize the treatment protocol and irradiate the mice when the anti-HER2 gold nanoshells had accumulated most in the tumors. After weekly injections of anti-HER2 gold nanoshells, and repeated irradiations with a femtosecond-pulsed laser over four weeks, tumor growth was significantly inhibited. Detailed tissue microscopic analyses showed that the tumor growth inhibition was due to an anti-angiogenic effect, coherent with a preferential distribution of the nanoshells in tumor microvessels. We also showed a direct tumor cell effect with apoptosis and inhibition of proliferation, coherent with an immune-mediated targeting of tumor cells by anti-HER2 nanoshells. CONCLUSION: This preclinical study thus supports the use of anti-HER2 gold nanoshells and photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer.

A successful compartmental approach for the treatment of breast cancer brain metastases.

BACKGROUND: Brain metastases are challenging daily practice in oncology and remain a compartmental problem since most anti-cancer drugs do not cross the blood-brain barrier at relevant pharmacological concentrations. METHODS: In a young woman with HER2-overexpressing breast cancer resistant to standard treatments, at the time of brain metastases progression, a ventricular reservoir was implanted for intrathecal drug injections and detailed pharmacokinetic studies. RESULTS: A first association of intrathecal trastuzumab with intravenous cisplatin was offered to the patient. For trastuzumab, the mean cerebrospinal fluid trough concentration of 53.4 mg/L reached relevant levels, enabling the stabilization of the metastases. Adding intravenous cisplatin was not beneficial, since the cerebrospinal fluid exposure was almost undetectable under 0.08 mg/L. We then offered the patient an intrathecal combination of trastuzumab and methotrexate, because of their in vitro synergic cytotoxicity. The cerebrospinal fluid peak of methotrexate was 1037 µmol/L at 2 h, and the concentrations remained above the theoretical therapeutic concentration. After 2 months of this drug combination, we obtained an excellent response on the brain metastases. CONCLUSION: Our preliminary study supports the interest of a compartmental approach through a direct administration of drugs into the cerebrospinal fluid for the treatment of breast cancer brain metastases.

Can trastuzumab emtansine be replaced by additional chemotherapy plus targeted therapy for HER2-overexpressing breast cancer patients with residual disease after neoadjuvant chemotherapy?

Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is an aggressive phenotype with a poor prognosis, and can easily metastasize and recur. Currently, chemotherapy plus HER2-targeted therapy is the standard systemic treatment for most of these patients. Given that neoadjuvant chemotherapy (NAC) has an efficacy equivalent to that of adjuvant chemotherapy and some additional benefits, many patients, especially those with more advanced tumors, prefer NAC and generally will not receive additional chemotherapy after surgery, irrespective of the pathological response. However, achieving pathological complete response to NAC is strongly correlated with prognosis, especially in triple-negative and HER2-overexpressing breast cancer. Therefore, postoperative treatment of these patients with residual diseases should be optimized to achieve favorable outcomes. The CREATE-X study has confirmed that additional chemotherapy can improve the outcomes of patients with HER2-negative residual disease after NAC. In addition, chemotherapy plays an indispensable role in the treatment of patients who receive surgery directly or who have recurrent lesions. Therefore, can additional chemotherapy improve prognosis of patients with HER2-overexpressing residual breast cancer? At present, no studies have compared the efficacy of additional chemotherapy plus trastuzumab with that of anti-HER2 therapy alone in residual cancer. The KATHERINE study revealed that trastuzumab emtansine (T-DM1) can reduce the risk of recurrence or death by 50% compared with trastuzumab in patients with HER2-positive residual invasive breast cancer after neoadjuvant therapy. T-DM1 is an antibody-drug conjugate of trastuzumab and the cytotoxic agent emtansine, and thus, to an extent, T-DM1 is equivalent to simultaneous application of chemotherapy and targeted therapy. However, high cost and low accessibility limit its use especially in low- and middle-income countries and regions. Hence, we proposed this perspective that additional chemotherapy plus trastuzumab should be given to HER2-overexpressing breast cancer patients with residual disease after NAC to improve their prognosis by discussing that the efficacy of additional chemotherapy plus trastuzumab is superior to that of anti-HER2 therapy alone and not inferior to T-DM1. Additional chemotherapy plus trastuzumab-based HER2-targeted therapy can be used as an alternative regimen to T-DM1 when T-DM1 is unavailable. However, further clinical research on the selection of chemotherapeutic agents is warranted.

Relationship between HER2 gene status and selected potential biological features related to trastuzumab resistance and its influence on survival of breast cancer patients undergoing trastuzumab adjuvant treatment.

BACKGROUND: The aim of the study was to investigate if parameters associated with human epidermal growth factor receptor type 2 (HER2) status (HER2 gene copy number, HER2/CEP17 ratio or polysomy of chromosome 17) are related to various biological features potentially responsible for trastuzumab resistance (PTEN, IGF-1R, MUC4, EGFR, HER3, HER4, and mutation status of PIK3CA) as well as their influence on survival of HER2-positive breast cancer patients treated with adjuvant chemotherapy and trastuzumab. PATIENTS AND METHODS: The investigated group consisted of 117 patients with invasive ductal breast cancer (T≥1, N≥0, M0) with overexpression of HER2, who underwent radical surgery between 2007 and 2014. Status of ER, PR, and HER2 expression was retrieved from patients' files. HER2 gene copy number was investigated by fluorescence in situ hybridization using PathVysion HER-2 DNA Probe Kit II. Expression of PTEN, IGF-1R, MUC4, EGFR, HER3, and HER4 was assessed immunohistochemically on formalin-fixed paraffin-embedded tissue sections. PIK3C mutation status was determined by qPCR analysis. RESULTS: Overexpression of HER2 protein (IHC 3+) and ER negativity corresponded to higher HER22 copy number and HER2/CEP17 ratio (.<0.001). Tumors with polysomy were characterized by higher HER22 gene copy number but lower HER2/CEP17p ratio (p<0.026, p<0.001). Patients with tumors featuring HER3 immunonegativity or low HER2/CEP17 ratio (#4) were characterized by 100% metastasis-free survival (.=0.018, p=0.062). CONCLUSION: Presence of both unfavorable factors, ie, HER3 expression and high HER2/CEP17 ratio, allowed to distinguish a group of patients with worse prognosis (.=0.001).

Micromolecular methods for diagnosis and therapeutic strategy: a case study.

An intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffin-embedding of the sample. Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We laser-microdissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2-overexpressing breast cancer cell line BT-474. We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy.

Proteins Involved in HER2 Signalling Pathway, Their Relations and Influence on Metastasis-Free Survival in HER2-Positive Breast Cancer Patients Treated with Trastuzumab in Adjuvant Setting.

AIM: Resistance to trastuzumab (which is a standard therapy for breast cancer patients with HER2 overexpression) is associated with higher risk of progression or cancer death, and might be related to activation of signalling cascades (PI3K/AKT/mTOR, Ras/Raf/MAPK) and decreased level of their inhibitors. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded tumour specimens from 118 HER2-overexpressing breast cancer patients treated with radical local therapy and trastuzumab in adjuvant setting were used for the assessment of: (1) PIK3CA gene mutations (p.H1047R and p.E545K) by qPCR, and (2) expression of Ki-67, EGFR, MUC4, HER3 and PTEN by immunohistochemistry. RESULTS: Lower Ki-67LI was observed in EGFR-immunonegative and in PTEN-immunopositive tumours. MUC4-immunonegative tumours more frequently were PTEN- and HER3-immunonegative. Favourable metastasis-free survival was observed in patients with tumours characterized by Ki-67LI≤50% (p=0.027), HER3 immunonegativity or PTEN immunopositivity (vs. tumours with HER3 expression and lack of PTEN expression, p=0.043), additionally, the trend was observed for patients with pN0+pN1 pathological tumour stage (vs. pN2+pN3) (p=0.086). Cox model revealed that independent negative prognostic factors were: (i) Ki-67LI>50% (p=0.014, RR=4.6, 95% CI 1.4-15.4), (ii) HER3 immunopositivity together with PTEN immunonegativity (p=0.034, RR=3.7, 95% CI 1.1-12.5). CONCLUSION: The results of our study suggest that combined analysis of HER3 and PTEN expression might bring information on trastuzumab sensitivity in the group of HER2-positive breast cancer patients treated with trastuzumab in adjuvant setting.

Neratinib for the treatment of breast cancer.

INTRODUCTION: Neratinib is an orally available, pan-HER inhibitor with clinical activity in patients with HER2-amplified and HER2-mutated breast cancer. Areas covered: A summary of publically available and relevant clinical data on neratinib. Expert opinion: Neratinib (N) is clearly distinct from lapatinib (L), a difference based on its broad anti-HER effect, its covalent target binding and its toxicity profile. The main toxicity of neratinib is gastro-intestinal and is essentially limited to diarrhea. Although not directly compared with single agent lapatinib, skin toxicity is much less pronounced with N. The direct clinical comparison of N-capecitabine versus L-capecitabine is the subject of the ongoing NALA-trial. In patients with advanced disease, neratinib has clinically relevant activity in patients with trastuzumab(T)-pretreated and unpretreated disease. In patients having completed one year of adjuvant trastuzumab, an additional year of neratinib further reduces the risk of recurrence of invasive disease. The activity of neratinib in HER2-mutated advanced disease is subject of ongoing clinical trials but preclinical and early clinical results are promising. Neratinib is a usefull drug and a valuable addition to the different anti-HER2-drugs avalaible for patients with HER2-overexpressing and HER2-mutated breast cancer.

Better predictive value of axillary lymph node (ALN) status after systemic therapy for operable HER2-overexpressing breast cancer: A single-institution retrospective study.

BACKGROUND: Thresholds for using Preoperative Systemic Therapy (PreST) have decreased to include early breast cancer. This study investigates the predictive value of axillary lymph node (ALN) status before and after systemic therapy and discusses whether it is better to receive PreST first in operable HER2-overexpressing breast cancer patients. METHODS: From January 2008 to June 2013 at Fudan University Shanghai Cancer Center, we identified 406 eligible female patients with stage II-IIIa, operable and pathologically confirmed HER2-overexpressing invasive ductal carcinoma. Of these patients, 269 underwent surgery first followed by chemotherapy plus trastuzumab (chemo-trastuzumab) (SurgFirst group), whereas 137 received systemic chemo-trastuzumab therapy first followed by surgery (STFirst group). Disease-free survival (DFS) and overall survival (OS) were evaluated according to different ALN statuses using the Kaplan-Meier method. Multivariate COX model analyses were also conducted. RESULTS: The median follow-up time was 47 months (IQR: 37-60). Both ALN status and overall pathological complete remission (pCR) status were shown to be significant for the prediction of DFS (p = 0.001 and p = 0.005, respectively) and OS (p = 0.009 and p = 0.027, respectively) in the STFirst group. However, patients with positive ALN(s) did not experience significantly poorer survival compared with those with negative ALN in the SurgFirst group. The adjusted hazard ratios (HRs) for positive ALN status in the STFirst and SurgFirst groups were 6.66 (p = 0.001, 95%CI: 2.18-20.38) and 2.40 (p = 0.126, 95%CI: 0.78-7.34), respectively. CONCLUSION: The ALN status after systemic chemo-trastuzumab therapy better predicts the survival outcome. We recommend the application of PreST followed by surgery in patients with operable HER2-overexpressing breast cancer.

Aloe-emodin inhibits HER-2 expression through the downregulation of Y-box binding protein-1 in HER-2-overexpressing human breast cancer cells.

Human epidermal growth factor receptor-2 (HER-2)-positive breast cancer tends to be aggressive, highly metastatic, and drug resistant and spreads rapidly. Studies have indicated that emodin inhibits HER-2 expression. This study compared the HER-2-inhibitory effects of two compounds extracted from rhubarb roots: aloe-emodin (AE) and rhein. Our results indicated that AE exerted the most potent inhibitory effect on HER-2 expression. Treatment of HER-2-overexpressing breast cancer cells with AE reduced tumor initiation, cell migration, and cell invasion. AE was able to suppress YB-1 expression, further suppressing downstream HER-2 expression. AE suppressed YB-1 expression through the inhibition of Twist in HER-2-overexpressing breast cancer cells. Our data also found that AE inhibited cancer metastasis and cancer stem cells through the inhibition of EMT. Interestingly, AE suppressed YB-1 expression through the downregulation of the intracellular integrin-linked kinase (ILK)/protein kinase B (Akt)/mTOR signaling pathway in HER-2-overexpressing breast cancer cells. In vivo study showed the positive result of antitumor activity of AE in nude mice injected with human HER-2-overexpressing breast cancer cells. These findings suggest the possible application of AE in the treatment of HER-2-positive breast cancer.

A non-randomized dose-escalation Phase I trial of a protein-based immunotherapeutic for the treatment of breast cancer patients with HER2-overexpressing tumors.

This Phase I dose-escalation study (NCT00058526) assessed the safety and immunogenicity of an anti-cancer immunotherapeutic (recombinant HER2 protein (dHER2) combined with the immunostimulant AS15) in patients with early-stage HER2-overexpressing breast cancer (BC). Sixty-one trastuzumab-naive patients with stage II-III HER2-positive BC received the dHER2 immunotherapeutic after surgical resection and adjuvant therapy. They were allocated into four cohorts receiving different doses of dHER2 (20, 100, 500 µg) combined with a fixed AS15 dose. Safety and immunogenicity (dHER2-specific antibody responses) were assessed. After completing the immunization schedule (three or six doses over 14 weeks) and a six-month follow-up, the patients were followed for 5 years for late toxicity, long-term immunogenicity, and clinical status. The immunizations were well tolerated, and increasing doses of dHER2 had no impact on the frequency or severity of adverse events. Few late toxicities were reported, and after 5 years 45/54 patients (83.3 %) were still alive, while 28/45 (62 %) with known disease status were disease free. Regarding the immunogenicity of the compound, a positive association was found between the dHER2 dose, the immunization schedule, and the prevalence of dHER2-specific humoral responses. Among the patients receiving the most intense immunization schedule with the highest dHER2 dose, 6/8 maintained their dHER2-specific antibody response 5 years after immunization. The dHER2 immunotherapeutic had an acceptable safety profile in early HER2-positive BC patients. dHER2-specific antibody responses were induced, with the rate of responders increasing with the dHER2 dose and the number and frequency of immunizations.

Neural Stem Cells Secreting Anti-HER2 Antibody Improve Survival in a Preclinical Model of HER2 Overexpressing Breast Cancer Brain Metastases.

The treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer has been revolutionized by trastuzumab. However, longer survival of these patients now predisposes them to forming HER2 positive brain metastases, as the therapeutic antibodies cannot cross the blood brain barrier. The current oncologic repertoire does not offer a rational, nontoxic targeted therapy for brain metastases. In this study, we used an established human neural stem cell line, HB1.F3 NSCs and generated a stable pool of cells secreting a high amount of functional full-length anti-HER2 antibody, equivalent to trastuzumab. Anti-HER2Ab secreted by the NSCs (HER2Ab-NSCs) specifically binds to HER2 overexpressing human breast cancer cells and inhibits PI3K-Akt signaling. This translates to HER2Ab-NSC inhibition of breast cancer cell growth in vitro. Preclinical in vivo experiments using HER2Ab overexpressing NSCs in a breast cancer brain metastases (BCBM) mouse model demonstrate that intracranial injection of HER2Ab-NSCs significantly improves survival. In effect, these NSCs provide tumor localized production of HER2Ab, minimizing any potential off-target side effects. Our results establish HER2Ab-NSCs as a novel, nontoxic, and rational therapeutic approach for the successful treatment of HER2 overexpressing BCBM, which now warrants further preclinical and clinical investigation.

Combating HER2-overexpressing breast cancer through induction of calreticulin exposure by Tras-Permut CrossMab.

Although trastuzumab has succeeded in breast cancer treatment, acquired resistance is one of the prime obstacles for breast cancer therapies. There is an urgent need to develop novel HER2 antibodies against trastuzumab resistance. Here, we first rational designed avidity-imporved trastuzumab and pertuzumab variants, and explored the correlation between the binding avidity improvement and their antitumor activities. After characterization of a pertuzumab variant L56TY with potent antitumor activities, a bispecific immunoglobulin G-like CrossMab (Tras-Permut CrossMab) was generated from trastuzumab and binding avidity-improved pertuzumab variant L56TY. Although, the antitumor efficacy of trastuzumab was not enhanced by improving its binding avidity, binding avidity improvement could significantly increase the anti-proliferative and antibody-dependent cellular cytotoxicity (ADCC) activities of pertuzumab. Further studies showed that Tras-Permut CrossMab exhibited exceptional high efficiency to inhibit the progression of trastuzumab-resistant breast cancer. Notably, we found that calreticulin (CRT) exposure induced by Tras-Permut CrossMab was essential for induction of tumor-specific T cell immunity against tumor recurrence. These data indicated that simultaneous blockade of HER2 protein by Tras-Permut CrossMab could trigger CRT exposure and subsequently induce potent tumor-specific T cell immunity, suggesting it could be a promising therapeutic strategy against trastuzumab resistance.