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Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked (i.e., supplemented) into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases.

Background: Neoadjuvant radiotherapy (naRT) in addition to neoadjuvant chemotherapy (naCT) has been used for locally advanced, inoperable breast cancer or to allow breast conserving surgery (BCS). Retrospective analyses suggest that naRT + naCT might result in an improvement in pathological complete response (pCR rate and disease-free survival). pCR is a surrogate parameter for improved event-free and overall survival (OS) and allows for the adaption of the post-neoadjuvant therapy regimens. However, it is not clear whether pCR achieved with the addition of naRT has the same prognostic value., Patients and Methods: We performed a retrospective re-analysis of 356 patients (cT1-cT4/cN0-N+) treated with naRT and naCT with a long-term follow-up. Patients underwent naRT on the breast and regional lymph nodes combined with a boost to the primary tumor. Chemotherapy with different agents was given either sequentially or concomitantly to naRT. We used the Cox proportional hazard regression model to estimate the effect of pCR in our cohort in different subgroups as well as chemotherapy protocols. Clinical response markers correlating with OS were also analyzed., Results: For patients with median follow-ups of 20 years, 10 years, 15 years, 20 years, and 25 years, OS rates were 69.7%, 60.6%, 53.1%, and 45.1%, respectively. pCR was achieved in 31.1% of patients and associated with a significant improvement in OS (HR = 0.58; CI-95%: 0.41-0.80; p = 0.001). The prognostic impact of pCR was evident across breast cancer subtypes and chemotherapy regimens. Multivariate analysis showed that age, clinical tumor and nodal stage, chemotherapy, and pCR were prognostic for OS., Conclusion: NaCT and naRT prior to surgical resection achieve good long-term survival in high-risk breast cancer. pCR after naRT maintains its prognostic value in breast cancer subtypes and across different subgroups. pCR driven by naRT and naCT independently influences long-term survival.

The prognostic relevance of circulating tumor cells (CTCs) in breast cancer is well established. However, little is known about the association of CTCs and site of first metastasis. In the SUCCESS A trial, 373 out of 3754 randomized high-risk breast cancer patients developed metastatic disease. CTC status was assessed by the FDA-approved CellSearch ® -System (Menarini Silicon Biosystems, Bologna, Italy) in 206 of these patients before chemotherapy and additionally in 159 patients after chemotherapy. CTCs were detected in 70 (34.0%) of 206 patients before (median 2 CTCs, 1-827) and in 44 (27.7%) of 159 patients after chemotherapy (median 1 CTC, 1-124); 16 (10.1%) of 159 patients were CTC-positive at both timepoints. The site of first distant disease was bone-only, visceral-only, and other-site-only in 44 (21.4%), 60 (29.1%), and 74 (35.9%) patients, respectively, while 28 (13.6%) patients had multiple sites of first metastatic disease. Patients with CTCs at both timepoints more often showed bone-only first distant disease (37.5% vs. 21.0%) and first distant disease at multiple sites (31.3% vs. 12.6%) than patients without CTCs before and/or after chemotherapy ( p = 0.027). In conclusion, the presence of CTCs before and after chemotherapy is associated with multiple-site or bone-only first-distant disease and may trigger intensified follow-up and perhaps further treatment.

Background/Aims: Due to its favorable dose distribution and targeting of the region at highest risk of recurrence due to direct visualization of tumor bed, intraoperative electron radiation therapy (IOERT) is used as part of a breast-conserving treatment approach. The aim of this study was to analyze tumor control and survival, as well as the toxicity profile, and cosmetic outcomes in patients irradiated with an IOERT boost for breast cancer. Materials and Methods: 139 Patients treated at our institution between January 2010 and January 2015 with a single boost dose of 10 Gy to the tumor bed during breast-conserving surgery followed by whole-breast irradiation were retrospectively analyzed. Results: 139 patients were included in this analysis. The median age was 54 years (range 28−83 years). The preferred surgical strategy was segmental resection with sentinel lymphonodectomy (66.5%) or axillary dissection (23.1%). Regarding adjuvant radiotherapy, the vast majority received 5 × 1.8 Gy to 50.4 Gy. At a median follow-up of 33.6 months, recurrence-free and overall survival were 95.5% and 94.9%, respectively. No patient developed an in-field recurrence. Seven patients (5.0%) died during the follow-up period, including two patients due to disease recurrence (non-in-field). High-grade (CTCAE > 2) perioperative adverse events attributable to IOERT included wound healing disorder (N = 1) and hematoma (N = 1). High-grade late adverse events (LENT-SOMA grade III) were reported only in one patient with fat necrosis. Low-grade late adverse events (LENT-SOMA grade I-II) included pain (18.0%), edema (10.5%), fibrosis (21%), telangiectasia (4.5%) and pigmentation change (23.0%). The mean breast retraction assessment score was 1.66 (0−6). Both patients and specialists rated the cosmetic result “excellent/good” in 84.8% and 87.9%, respectively. Conclusion: Our study reports favorable data on the cosmetic outcome as well as the acute and early long-term tolerability for patients treated with an IOERT boost. Our oncologic control rates are comparable to the previous literature. However, prospective investigations on the role of IOERT in comparison to other boost procedures would be desirable.

Patients with high-risk non-metastatic breast cancer are recommended for chemotherapy, preferably in the neoadjuvant setting. Beyond advantages such as a better operability and an improved assessment of individual prognosis, the preoperative administration of systemic treatment offers the unique possibility of selecting postoperative therapies according to tumor response. In patients with HER2-positive disease, both the escalation of therapy in the case of high-risk features and the de-escalation in patients with a low tumor load are currently discussed. Patients with small node-negative tumors receive primary surgery and, upon confirmation of pathological T1 N0 status, de-escalated adjuvant therapy with paclitaxel and trastuzumab. For those with a large tumor and/or nodal involvement, neoadjuvant polychemotherapy with a dual antibody blockade is recommended. Patients with invasive residual disease benefit from switching postoperative therapy to the antibody-drug-conjugate trastuzumab emtansine (T-DM1). In this review, we discuss current evidence and controversies regarding post-neoadjuvant treatment strategies in HER2-positive breast cancer.

Neoadjuvant chemotherapy enables close monitoring of tumor response in patients with breast cancer. Being able to assess tumor response during treatment provides an opportunity to evaluate new therapeutic strategies. Thus, for triple-negative breast tumors, it was demonstrated that additional immunotherapy could improve prognosis compared with chemotherapy alone. Furthermore, adjuvant therapy can be escalated or de-escalated correspondingly. The CREATE-X trial randomly assigned HER2-negative patients with residual tumor after neoadjuvant therapy to either observation or capecitabine. In HER2-negative patients with positive BRCA testing, the OlympiA study randomly assigned patients to either observation or olaparib. HER2-positive patients without pathologic remission were randomly assigned to trastuzumab or trastuzumab-emtansine within the KATHERINE study. These studies were all able to show an improvement in oncologic outcome associated with the escalation of therapy in patients presenting with residual tumor after neoadjuvant treatment. On the other hand, this individualization of therapy may also offer the possibility to de-escalate treatment, and thereby reduce morbidity. Among WSG-ADAPT HER2+/HR-, HER2-positive patients achieved comparable results without chemotherapy after complete remission following neoadjuvant treatment. In summary, the concept of post-neoadjuvant therapy constitutes a great opportunity for individualized cancer treatment, potentially improving outcome. In this review, the most important trials of post-neoadjuvant therapy are compiled and discussed.

Background: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof-of-concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients., Methods: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs., Results: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient's CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging., Conclusion: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens.

In previous studies, we reported that progesterone receptor membrane component 1 (PGRMC1) is implicated in progestin signaling and possibly associated with increased breast cancer risk upon combined hormone replacement therapy. To gain mechanistic insight, we searched for potential PGRMC1 interaction partners upon progestin treatment by co-immunoprecipitation and mass spectrometry. The interactions with the identified partners were further characterized with respect to PGRMC1 phosphorylation status and with emphasis on the crosstalk between PGRMC1 and estrogen receptor α (ERα). We report that PGRMC1 overexpression resulted in increased proliferation of hormone receptor positive breast cancer cell lines upon treatment with a subgroup of progestins including norethisterone and dydrogesterone that promote PGRMC1-phosphorylation on S181. The ERα modulators prohibitin-1 (PHB1) and prohibitin-2 (PHB2) interact with PGRMC1 in dependency on S181-phosphorylation upon treatment with the same progestins. Moreover, increased interaction between PGRMC1 and PHBs correlated with decreased binding of PHBs to ERα and subsequent ERα activation. Inhibition of either PGRMC1 or ERα abolished this effect. In summary, we provide strong evidence that activated PGRMC1 associates with PHBs, competitively removing them from ERα, which then can develop its transcriptional activities on target genes. This study emphasizes the role of PGRMC1 in a key breast cancer signaling pathway which may provide a new avenue to target hormone-dependent breast cancer.

Purpose: Post-treatment follow-up in women with cervical pre-cancers (CIN3) is mandatory due to relapse in up to 10% of patients. Standard follow-up based on hrHPV-DNA/cytology co-testing has high sensitivity but limited specificity. The aim of our prospective, multicenter, observational study was to test the hypothesis that an individualized viral-cellular-junction test (vcj-PCR) combined with cytology has a lower false positive rate for the prediction of recurrence compared to standard co-testing., Methods: Pre-surgical cervical swabs served for the identification of HPV16/18 DNA integration sites by next-generation-sequencing (NGS). Samples taken at 6, 12 and 24 months post-surgery were evaluated by cytology, hrHPV-DNA and the patients' individual HPV-integration sites (vcj-PCR on the basis of NGS)., Results: Integration sites were detected in 48 of 445 patients (10.8%), 39 of them had valid follow-up data. The false positive rate was 18.2% (95% CI 8.6-34.4%) for standard hrHPV/cytology at six months compared to 12.1% (95% CI 4.8-27.3%) for vcj-PCR/cytology, respectively (McNemar p = 0.50). Six patients developed recurrences (1 CIN2, 5 CIN3) during follow-up. Standard co-testing detected all, whereas vcj-PCR/cytology detected only five patients with recurrences. Data of 269 patients without evidence of HPV16/18 integration were subject to post-hoc analyses. Standard co-testing revealed a false positive rate of 15.7% (95% CI 11.7-20.7%) and predicted ten of fourteen recurrences at six months., Conclusions: Although highly specific on its own vcj-PCR could not detect all recurrent CIN2/3. Possible reasons for this unexpected result may be multifocal lesions, intratumoral heterogeneity with respect to HPV integration and/or incident CIN.

Several scores have been developed in order to estimate the prognosis of patients with brain metastases (BM) by objective criteria. The aim of this analysis was to validate all three published graded-prognostic-assessment (GPA)-scores in a subcohort of 882 breast cancer (BC) patients with BM in the Brain Metastases in the German Breast Cancer (BMBC) registry. The median age at diagnosis of BM was 57 years. All in all, 22.3% of patients ( n = 197) had triple-negative, 33.4% ( n = 295) luminal A like, 25.1% ( n = 221) luminal B/HER2-enriched like and 19.2% ( n = 169) HER2 positive like BC. Age ≥60 years, evidence of extracranial metastases (ECM), higher number of BM, triple-negative subtype and low Karnofsky-Performance-Status (KPS) were all associated with worse overall survival (OS) in univariate analysis ( p < 0.001 each). All three GPA-scores were associated with OS. The breast-GPA showed the highest probability of classifying patients with survival above 12 months in the best prognostic group (specificity 68.7% compared with 48.1% for the updated breast-GPA and 21.8% for the original GPA). Sensitivities for predicting 3 months survival were very low for all scores. In this analysis, all GPA-scores showed only moderate diagnostic accuracy in predicting the OS of BC patients with BM.

Background : Brain metastases (BM) have become a major challenge in patients with metastatic breast cancer. Methods : The aim of this analysis was to characterize patients with asymptomatic BM ( n = 580) in the overall cohort of 2589 patients with BM from our Brain Metastases in Breast Cancer Network Germany (BMBC) registry. Results : Compared to symptomatic patients, asymptomatic patients were slightly younger at diagnosis (median age: 55.5 vs. 57.0 years, p = 0.01), had a better performance status at diagnosis (Karnofsky index 80-100%: 68.4% vs. 57%, p < 0.001), a lower number of BM (>1 BM: 56% vs. 70%, p = 0.027), and a slightly smaller diameter of BM (median: 1.5 vs. 2.2 cm, p < 0.001). Asymptomatic patients were more likely to have extracranial metastases (86.7% vs. 81.5%, p = 0.003) but were less likely to have leptomeningeal metastasis (6.3% vs. 10.9%, p < 0.001). Asymptomatic patients underwent less intensive BM therapy but had a longer median overall survival (statistically significant for a cohort of HER2-positive patients) compared to symptomatic patients (10.4 vs. 6.9 months, p < 0.001). Conclusions : These analyses show a trend that asymptomatic patients have less severe metastatic brain disease and despite less intensive local BM therapy still have a better outcome (statistically significant for a cohort of HER2-positive patients) than patients who present with symptomatic BM, although a lead time bias of the earlier diagnosis cannot be ruled out. Our analysis is of clinical relevance in the context of potential trials examining the benefit of early detection and treatment of BM.

Evidence suggests that the DNA end-binding protein p53-binding protein 1 (53BP1) is down-regulated in subsets of breast cancer. Circulating tumor cells (CTCs) provide accessible "biopsy material" to track cell traits and functions and their alterations during treatment. Here, we prospectively monitored the 53BP1 status in CTCs from 67 metastatic breast cancer (MBC) patients with HER2- CTCs and known hormone receptor (HR) status of the primary tumor and/or metastases before, during, and at the end of chemotherapeutic treatment with Eribulin. Nuclear 53BP1 staining and genomic integrity were evaluated by immunocytochemical and whole-genome-amplification-based polymerase chain reaction (PCR) analysis, respectively. Comparative analysis of CTCs from patients with triple-negative and HR+ tumors revealed elevated 53BP1 levels in CTCs from patients with HR+ metastases, particularly following chemotherapeutic treatment. Differences in nuclear 53BP1 signals did not correlate with genomic integrity in CTCs at baseline or with nuclear γH2AX signals in MBC cell lines, indicating that 53BP1 detected features beyond DNA damage. Kaplan-Meier analysis revealed an increasing association between nuclear 53BP1-positivity and progression-free survival (PFS) during chemotherapy until the final visit. Our data suggest that 53BP1 detection in CTCs could be a useful marker to capture dynamic changes of chemotherapeutic responsiveness in triple-negative and HR+ MBC.

Circulating tumor cells (CTCs) hold great promise with regard to prognosis, treatment optimization, and monitoring of breast cancer patients. Single CTC transcriptome profiling might help reveal valuable information concerning intra-patient heterogeneity relevant to therapeutic interventions. In this study, we combined Diagnostic Leukapheresis (DLA), which is a microfluidic enrichment using the Parsortix TM system, micromanipulation with CellCelector TM and subsequent single cell multi-marker transcriptome profiling. First, a PCR panel consisting of 30 different endocrine resistance and phenotypic marker genes was validated for single cell profiling by using different breast cancer cell lines. Second, this panel was applied to characterize uncultured and cultured CTCs, which were enriched from a cryopreserved DLA product obtained from a patient suffering from metastatic breast cancer resistant to endocrine therapy. Gene expression profiles of both CTC populations uncovered inter CTC heterogeneity for transcripts, which are associated with response or resistance to endocrine therapy (e.g., ESR1, HER2, FGFR1 ). Hierarchical clustering revealed CTC subpopulations with different expressions of transcripts regarding the CTCs' differential phenotypes ( EpCAM, CD44, CD24, MYC, MUC1 ) and of transcripts involved in endocrine signaling pathways ( FOXO, PTEN ). Moreover, ER-positive CTCs exhibited significant higher expression of Cyclin D1, which might be relevant for CDK4/6 inhibitor therapies. Overall, gene expression profiles of uncultured and cultured CTCs resulted in a partly combined grouping. Our findings demonstrate that multi-marker RNA profiling of enriched single uncultured CTCs and cultured CTCs form cryopreserved DLA samples may provide important insights into intra-patient heterogeneity relevant for targeted therapies and therapy resistance.

Tumor cell dissemination is a common phenomenon observed in most cancers of epithelial origin. One-third of breast cancer patients present with disseminated tumor cells (DTCs) in bone marrow at time of diagnosis; these patients, as well as patients with persistent DTCs, have significantly worse clinical outcome than DTC-negative patients. Since DTC phenotype may differ from the primary tumor with regard to ER and HER2 status, reevaluation of predictive markers on DTCs may optimize treatment choices. In the present review, we report on the clinical relevance of DTC detection in breast cancer.