Characterization of Hormone Receptor and HER2 Status in Breast Cancer Using Mass Spectrometry Imaging.

Immunohistochemical evaluation of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 status stratify the different subtypes of breast cancer and define the treatment course. Triple-negative breast cancer (TNBC), which does not register receptor overexpression, is often associated with worse patient prognosis. Mass spectrometry imaging transcribes the molecular content of tissue specimens without requiring additional tags or preliminary analysis of the samples, being therefore an excellent methodology for an unbiased determination of tissue constituents, in particular tumor markers. In this study, the proteomic content of 1191 human breast cancer samples was characterized by mass spectrometry imaging and the epithelial regions were employed to train and test machine-learning models to characterize the individual receptor status and to classify TNBC. The classification models presented yielded high accuracies for estrogen and progesterone receptors and over 95% accuracy for classification of TNBC. Analysis of the molecular features revealed that vimentin overexpression is associated with TNBC, supported by immunohistochemistry validation, revealing a new potential target for diagnosis and treatment.

EVI1 expression in early-stage breast cancer patients treated with neoadjuvant chemotherapy.

BACKGROUND: Overexpression of the EVI1 (ecotropic viral integration site 1) oncogene has recently been implicated as a prognostic factor in breast cancer (BC), particularly in triple-negative BC (TNBC). In this study we aimed to investigate frequency and clinical relevance of EVI1 expression in newly diagnosed BC treated with neoadjuvant chemotherapy. METHODS: EVI1 expression was determined by immunohistochemistry using H-score as a cumulative measurement of protein expression in pretherapeutic biopsies of BC patients treated with anthracycline/taxane based neoadjuvant chemotherapy within the GeparTrio trial. EVI1 was analyzed as a continuous variable and dichotomized into low or high based on median expression. Endpoints were pathological complete response (pCR), disease-free survival (DFS) and overall survival (OS). RESULTS: Of the 993 tumors analyzed, 882 had available subtype information: 50.8% were HR + /HER2-, 15% HR + /HER2 + , 9.8% HR-/HER2 + , and 24.5% TNBC. Median EVI1 H-score was 112.16 (range 0.5-291.4). High EVI1 expression was significantly associated with smaller tumor size (p = 0.002) but not with BC subtype. Elevated EVI1 levels were not significantly associated with therapy response and survival in the entire cohort or within BC subtypes. However, TNBC patients with high EVI1 showed a trend towards increased pCR rates compared to low group (37.7% vs 27.5%, p = 0.114; odds ratio 1.60 (95%CI 0.90-2.85, p = 0.110) and numerically better DFS (HR = 0.77 [95%CI 0.48-1.23], log-rank p = 0.271) and OS (HR = 0.76 [95% 0.44-1.31], log-rank p = 0.314) without reaching statistical significance. CONCLUSION: EVI1 was not associated with response to neoadjuvant therapy or patient survival in the overall cohort. Further analyses are needed to verify our findings especially in the pathological work-up of early-stage HER2-negative BC patients. TRIAL REGISTRATION: NCT00544765.

Fixed-dose combination of pertuzumab and trastuzumab for subcutaneous injection plus chemotherapy in HER2-positive early breast cancer (FeDeriCa): a randomised, open-label, multicentre, non-inferiority, phase 3 study.

BACKGROUND: A subcutaneous formulation of pertuzumab and trastuzumab with recombinant human hyaluronidase in one ready-to-use, fixed-dose combination vial (pertuzumab, trastuzumab, and hyaluronidase-zzxf) was approved by the US Food and Drug Administration (FDA) on June 29, 2020. We report the primary analysis of the FeDeriCa study, which was designed to assess the pharmacokinetics, efficacy, and safety of the fixed-dose subcutaneous formulation compared to intravenous pertuzumab plus trastuzumab in patients with HER2-positive early breast cancer in the neoadjuvant-adjuvant setting. METHODS: FeDeriCa, a randomised, open-label, international, multicentre, non-inferiority, phase 3 study, was done across 106 sites in 19 countries. Patients aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0 or 1, HER2-positive, operable, locally advanced, or inflammatory stage II-IIIC breast cancer, and a left ventricular ejection fraction of 55% or more were randomly assigned (1:1), using a voice-based or web-based response system, to receive intravenous pertuzumab (840 mg loading dose, followed by 420 mg maintenance doses) plus intravenous trastuzumab (8 mg/kg loading dose, followed by 6 mg/kg maintenance doses) or the fixed-dose combination of pertuzumab and trastuzumab for subcutaneous injection (1200 mg pertuzumab plus 600 mg trastuzumab loading dose in 15 mL, followed by 600 mg pertuzumab plus 600 mg trastuzumab maintenance doses in 10 mL), both administered every 3 weeks with neoadjuvant chemotherapy. Patients were stratified by hormone receptor status, clinical stage, and chemotherapy regimen. The investigator selected one of the two protocol-approved standard chemotherapy regimens before randomisation. Four cycles of HER2-targeted therapy were administered concurrently with the taxane. After surgery, patients continued the HER2-targeted therapy to receive an additional 14 cycles (total of 18). The primary endpoint was non-inferiority of the cycle 7 pertuzumab serum trough concentration (Ctrough; ie, cycle 8 predose pertuzumab concentration) within the fixed-dose combination for subcutaneous injection versus intravenous pertuzumab plus trastuzumab in the per-protocol pharmacokinetic population (all enrolled patients who adhered to prespecified criteria for pharmacokinetic assessment). Non-inferiority was concluded if the lower bound of the 90% CI of the geometric mean ratio was 0·8 or higher. The safety population included all patients who received at least one dose of study medication, including chemotherapy or HER2-targeted therapy. Enrolment, neoadjuvant therapy, and surgery have been completed; adjuvant treatment and follow-up are ongoing. The trial is registered with ClinicalTrials.gov, NCT03493854. FINDINGS: Between June 14, 2018, and Dec 24, 2018, 252 patients were randomly assigned to the intravenous infusion group and 248 to the fixed-dose combination group. The geometric mean ratio of pertuzumab serum Ctrough subcutaneous to serum Ctrough intravenous was 1·22 (90% CI 1·14-1·31). The most common grade 3-4 adverse events occurring during neoadjuvant treatment with HER2-targeted therapy plus chemotherapy in 5% or more of patients were neutropenia (34 [13%] of 252 patients in the intravenous infusion group vs 35 [14%] of 248 patients in the fixed-dose combination group), decreased neutrophil count (31 [12%] vs 27 [11%]), febrile neutropenia (14 [6%] vs 16 [6%]), diarrhoea (12 [5%] vs 17 [7%]), and decreased white blood cell count (18 [7%] vs nine [4%]). At least one treatment-related serious adverse event was reported in 25 (10%) patients in the intravenous infusion group and 26 (10%) patients in the fixed-dose combination group. One patient in each treatment group had an adverse event that led to death (urosepsis in the intravenous infusion group and acute myocardial infarction in the fixed-dose combination group); neither death was related to HER2-targeted therapy. INTERPRETATION: The study met its primary endpoint: the fixed-dose combination of pertuzumab and trastuzumab for subcutaneous injection provides non-inferior cycle 7 pertuzumab serum Ctrough concentrations to intravenous pertuzumab plus trastuzumab in the neoadjuvant setting with comparable total pathological complete response rates, supporting the FDA approval. Safety was similar between treatment groups, and in line with other pertuzumab, trastuzumab, and chemotherapy trials. Follow-up is ongoing for long-term outcomes, including efficacy and long-term safety. FUNDING: F Hoffmann-La Roche and Genentech.

TGFB-induced factor homeobox 1 (TGIF) expression in breast cancer.

BACKGROUND: Breast cancer (BC) is the most frequent female cancer and preferentially metastasizes to bone. The transcription factor TGFB-induced factor homeobox 1 (TGIF) is involved in bone metabolism. However, it is not yet known whether TGIF is associated with BC bone metastasis or patient outcome and thus of potential interest. METHODS: TGIF expression was analyzed by immunohistochemistry in 1197 formalin-fixed, paraffin-embedded tissue samples from BC patients treated in the GAIN (German Adjuvant Intergroup Node-Positive) study with two adjuvant dose-dense schedules of chemotherapy with or without bisphosphonate ibandronate. TGIF expression was categorized into negative/low and moderate/strong staining. Endpoints were disease-free survival (DFS), overall survival (OS) and time to primary bone metastasis as first site of relapse (TTPBM). RESULTS: We found associations of higher TGIF protein expression with smaller tumor size (p = 0.015), well differentiated phenotype (p < 0.001) and estrogen receptor (ER)-positive BC (p < 0.001). Patients with higher TGIF expression levels showed a significantly longer disease-free (DFS: HR 0.75 [95%CI 0.59-0.95], log-rank p = 0.019) and overall survival (OS: HR 0.69 [95%CI 0.50-0.94], log-rank p = 0.019), but no association with TTPBM (HR 0.77 [95%CI 0.51-1.16]; p = 0.213). Univariate analysis in molecular subgroups emphasized that elevated TGIF expression was prognostic for both DFS and OS in ER-positive BC patients (DFS: HR 0.68 [95%CI 0.51-0.91]; log-rank p = 0.009, interaction p = 0.130; OS: HR 0.60 [95%CI 0.41-0.88], log-rank p = 0.008, interaction p = 0.107) and in the HER2-negative subgroup (DFS:HR 0.67 [95%CI 0.50-0.88], log-rank p = 0.004, interaction p = 0.034; OS: HR 0.57 [95%CI 0.40-0.81], log-rank p = 0.002, interaction p = 0.015). CONCLUSIONS: Our results suggest that moderate to high TGIF expression is a common feature of breast cancer cells and that this is not associated with bone metastases as first site of relapse. However, a reduced expression is linked to tumor progression, especially in HER2-negative breast cancer. TRIAL REGISTRATION: This clinical trial has been registered with ClinicalTrials.gov ; registration number: NCT00196872 .

Dasatinib prevents skeletal metastasis of osteotropic MDA-MB-231 cells in a xenograft mouse model.

PURPOSE: Bone metastasis in breast cancer has been linked to activity of c-Src kinase, one of the extensively explored tyrosine kinases in cell biology. The impact of TNF-related apoptosis inducing ligand (TRAIL) and TRAIL receptors has just recently been integrated into this conception. METHODS: An osteotropic clone of MDA-MB-231 cells simulated a model for bone metastasis of triple-negative breast cancer (TNBC). The effects of Dasatinib, a clinically established inhibitor of Src kinases family and Abl were evaluated in vitro and in vivo. In vivo effects of Dasatinib treatment on the occurrence of skeletal metastases were tested in a xenograft mouse model after intra-cardiac injection of osteotropic MDA-MB-231-cells. Ex vivo analyses of the bone sections confirmed intraosseous growth of metastases and allowed determination of osteoclastic activity. RESULTS: Treatment of osteotropic MDA-MB-231 cells with Dasatinib inhibited proliferation rates in vitro. A shift in TRAIL-receptor expression towards an induction of oncogenic TRAIL-R2 was observed. In vivo, 15 of 30 mice received an intra-peritoneal treatment with Dasatinib. These mice showed significantly less skeletal metastases in bioluminescence scans. Moreover, a pronounced increase in bone volume was observed in the treatment group, as detected by µ-Computed Tomography. Dasatinib treatment also led to a greater increase in bone density in tibiae without metastatic affection, which was accompanied by reduced recruitment of osteoclasts. CONCLUSION: Our observations support the concept of utilizing Dasatinib in targeting early-stage bone metastatic TNBC and sustaining bone health.

Human leucocyte antigen class I in hormone receptor-positive, HER2-negative breast cancer: association with response and survival after neoadjuvant chemotherapy.

BACKGROUND: Clinical application of cancer immunotherapy requires a better understanding of tumor immunogenicity and the tumor microenvironment. HLA class I molecules present antigens to CD8+ cytotoxic cells. Their loss or downregulation is frequently found in tumors resulting in reduced T cell responses and worse prognosis. METHODS: We evaluated HLA class I heavy chain expression by immunohistochemistry in 863 biopsies (GeparTrio trial). Patients received neoadjuvant chemotherapy and adjuvant endocrine treatment if tumors were hormone receptor-positive (HR+). In parallel, the expression of HLA-A was analyzed using a microarray cohort of 320 breast cancer patients from the MD Anderson Cancer Center. We evaluated its association with clinical outcome, tumor-infiltrating lymphocytes (TILs), and immune cell metagenes. RESULTS: In HR+/HER2- breast cancer, HLA class I heavy chain expression was associated with increased TILs and better response to chemotherapy (7% vs. 14% pCR rate, P = 0.029), but worse disease-free survival (hazard ratio (HR) 1.6 (1.1-2.4); P = 0.024). The effect was significant in a multivariate model adjusted for clinical and pathological variables (HR 1.7 (1.1-2.6); P = 0.016) and was confirmed by analysis of HLA-A in a microarray cohort. HLA-A was correlated to most immune cell metagenes. There was no association with response or survival in triple-negative or HER2+ disease. CONCLUSIONS: The study confirms the negative prognostic role of lymphocytes in HR+ breast cancer and points at a complex interaction between chemotherapy, endocrine treatment, and tumor immunogenicity. The results point at a subtype-specific and potentially treatment-specific role of tumor-immunological processes in breast cancer with different implications in triple-negative and hormone receptor-positive disease.

Development of central nervous system metastases as a first site of metastatic disease in breast cancer patients treated in the neoadjuvant trials GeparQuinto and GeparSixto.

BACKGROUND: The incidence of central nervous system (CNS) metastases in breast cancer patients is rising and has become a major clinical challenge. Only few data are published concerning risk factors for the development of CNS metastases as a first site of metastatic disease in breast cancer patients. Moreover, the incidence of CNS metastases after modern neoadjuvant treatment is not clear. METHODS: We analyzed clinical factors associated with the occurrence of CNS metastases as the first site of metastatic disease in breast cancer patients after neoadjuvant treatment in the trials GeparQuinto and GeparSixto (n = 3160) where patients received targeted treatment in addition to taxane and anthracycline-based chemotherapy. RESULTS: After a median follow-up of 61 months, 108 (3%) of a total of 3160 patients developed CNS metastases as the first site of recurrence and 411 (13%) patients had metastatic disease outside the CNS. Thirty-six patients (1%) developed both CNS metastases and other distant metastases as the first site of metastatic disease. Regarding subtypes of the primary tumor, 1% of luminal A-like (11/954), 2% of luminal B-like (7/381), 4% of HER2-positive (34/809), and 6% of triple-negative patients (56/1008) developed CNS metastases as the first site of metastatic disease. In multivariate analysis, risk factors for the development of CNS metastases were larger tumor size (cT3-4; HR 1.63, 95% CI 1.08-2.46, p = 0.021), node-positive disease (HR 2.57, 95% CI 1.64-4.04, p < 0.001), no pCR after neoadjuvant chemotherapy (HR 2.29, 95% CI 1.32-3.97, p = 0.003), and HER2-positive (HR 3.80, 95% CI 1.89-7.64, p < 0.001) or triple-negative subtype (HR 6.38, 95% CI 3.28-12.44, p < 0.001). CONCLUSIONS: Especially patients with HER2-positive and triple-negative tumors are at risk of developing CNS metastases despite effective systemic treatment. A better understanding of the underlying mechanisms is required in order to develop potential preventive strategies.

Evaluation of soluble carbonic anhydrase IX as predictive marker for efficacy of bevacizumab: A biomarker analysis from the geparquinto phase III neoadjuvant breast cancer trial.

We analyzed the predictive potential of pretreatment soluble carbonic anhydrase IX levels (sCAIX) for the efficacy of bevacizumab in the phase III neoadjuvant GeparQuinto trial. sCAIX was determined by enzyme-linked immunosorbent assay (ELISA). Correlations between sCAIX and pathological complete response (pCR), disease-free and overall survival (DFS, OS) were assessed with logistic and Cox proportional hazard regression models using bootstrapping for robust estimates and internal validation. 1,160 HER2-negative patient sera were analyzed, of whom 577 received bevacizumab. Patients with low pretreatment sCAIX had decreased pCR rates (12.1 vs. 20.1%, p = 0.012) and poorer DFS (adjusted 5-year DFS 71.4 vs. 80.5 months, p = 0.010) compared to patients with high sCAIX when treated with neoadjuvant chemotherapy (NCT). For patients with low sCAIX, pCR rates significantly improved upon addition of bevacizumab to NCT (12.1 vs. 20.4%; p = 0.017), which was not the case in patients with high sCAIX (20.1% for NCT vs. 17.0% for NCT-B, p = 0.913). When analyzing DFS we found that bevacizumab improved 5-year DFS for patients with low sCAIX numerically but not significantly (71.4 vs. 78.5 months; log rank 0.234). In contrast, addition of bevacizumab worsened 5-year DFS for patients with high sCAIX (81 vs. 73.6 months, log-rank 0.025). By assessing sCAIX levels we identified a patient cohort in breast cancer that is potentially undertreated with NCT alone. Bevacizumab improved pCR rates in this group, suggesting sCAIX is a predictive biomarker for bevacizumab with regards to treatment response. Our data also show that bevacizumab is not beneficial in patients with high sCAIX.

Androgen receptor expression and response to chemotherapy in breast cancer patients treated in the neoadjuvant TECHNO and PREPARE trial.

BACKGROUND: The androgen receptor (AR) is discussed as a prognostic and/or predictive marker in breast cancer patients. METHODS: AR mRNA expression was analysed by RT-qPCR in breast cancer patients treated in the neoadjuvant TECHNO (n = 118, HER2-positive) and PREPARE trial (n = 321, HER2-positive and -negative). In addition, mRNA expression of the AR transcript variants 1 (AR1) and 2 (AR2) was measured. RESULTS: Regarding subtypes, high AR mRNA levels were frequent in HER2-positive (61.3%, 92/150) and luminal tumours (60.0%, 96/160) but almost absent in triple-negative tumours (4.3%, 3/69) (p < 0.0001). Overall, high AR mRNA levels were found to be associated with lower pathological complete remission (pCR) rates (OR 0.77 per unit, 95% CI 0.67-0.88, p = 0.0002) but also with better prognosis in terms of longer disease-free survival (DFS) (HR 0.57, 95% CI 0.39-0.85, p = 0.0054) and overall survival (OS) (HR 0.43, 95% CI, 0.26-0.71, p = 0.0011). In the PREPARE trial, a survival difference for patients with high and low AR1 mRNA levels could only be seen in the standard chemotherapy arm but not in the dose-dense treatment arm (OS: HR 0.41; 95% CI 0.22-0.74 vs. HR 1.05; 95% CI 0.52-2.13; p = 0.0459). CONCLUSIONS: We provide evidence that AR mRNA predicts response to chemotherapy in breast cancer patients.

Post-Mastectomy Radiotherapy After Neoadjuvant Chemotherapy in Breast Cancer: A Pooled Retrospective Analysis of Three Prospective Randomized Trials.

BACKGROUND: The impact of locoregional radiotherapy (RT) after neoadjuvant chemotherapy (NACT) and mastectomy in breast cancer patients is currently unclear. Several publications have suggested that patients with a favorable response to NACT might not benefit from RT after mastectomy. METHODS: A retrospective analysis of three prospective randomized NACT trials was performed. Information on the use of RT was available for 817 breast cancer patients with non-inflammatory breast cancer who underwent mastectomy after NACT within the GeparTrio, GeparQuattro, and GeparQuinto-trials. RT was administered to 676 of these patients (82.7%). RESULTS: The 5-year cumulative incidence of locoregional recurrence (LRR) was 15.2% (95% confidence interval [CI] 9.0-22.8%) in patients treated without RT and 11.3% in patients treated with RT (95% CI 8.7-14.3%). In the multivariate analysis, RT was associated with a lower risk of LRR (hazard ratio 0.51, 95% CI 0.27-1.0; p = 0.05). This effect was shown especially in patients with cT3/4 tumors, as well as in patients who were cN+ before neoadjuvant therapy, including those who converted to ypN0 after neoadjuvant therapy. In the bivariate analysis, disease-free survival was significantly worse in patients who received RT, however this was not confirmed in the multivariate analysis. CONCLUSIONS: Our results suggest that RT reduces the LRR rates in breast cancer patients who receive a mastectomy after NACT without an improvement in DFS. Prospective randomized controlled trials such as the National Surgical Adjuvant Breast and Bowel Project B-51/RTOG 1304 trial will analyze whether RT has any benefit in patients who have a favorable response after NACT.

Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are predictive for response to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) and HER2-positive breast cancer, but their role in luminal breast cancer and the effect of TILs on prognosis in all subtypes is less clear. Here, we assessed the relevance of TILs for chemotherapy response and prognosis in patients with TNBC, HER2-positive breast cancer, and luminal-HER2-negative breast cancer. METHODS: Patients with primary breast cancer who were treated with neoadjuvant combination chemotherapy were included from six randomised trials done by the German Breast Cancer Group. Pretherapeutic core biopsies from 3771 patients included in these studies were assessed for the number of stromal TILs by standardised methods according to the guidelines of the International TIL working group. TILs were analysed both as a continuous parameter and in three predefined groups of low (0-10% immune cells in stromal tissue within the tumour), intermediate (11-59%), and high TILs (≥60%). We used these data in univariable and multivariable statistical models to assess the association between TIL concentration and pathological complete response in all patients, and between the amount of TILs and disease-free survival and overall survival in 2560 patients from five of the six clinical trial cohorts. FINDINGS: In the luminal-HER2-negative breast cancer subtype, a pathological complete response (pCR) was achieved in 45 (6%) of 759 patients with low TILs, 48 (11%) of 435 with intermediate TILs, and 49 (28%) of 172 with high TILs. In the HER2-positive subtype, pCR was observed in 194 (32%) of 605 patients with low TILs, 198 (39%) of 512 with intermediate TILs, and 127 (48%) of 262 with high TILs. Finally, in the TNBC subtype, pCR was achieved in 80 (31%) of 260 patients with low TILs, 117 (31%) of 373 with intermediate TILs, and 136 (50%) of 273 with high TILs (p<0·0001 for each subtype, χ2 test for trend). In the univariable analysis, a 10% increase in TILs was associated with longer disease-free survival in TNBC (hazard ratio [HR] 0·93 [95% CI 0·87-0·98], p=0·011) and HER2-positive breast cancer (0·94 [0·89-0·99], p=0·017), but not in luminal-HER2-negative tumours (1·02 [0·96-1·09], p=0·46). The increase in TILs was also associated with longer overall survival in TNBC (0·92 [0·86-0·99], p=0·032), but had no association in HER2-positive breast cancer (0·94 [0·86-1·02], p=0·11), and was associated with shorter overall survival in luminal-HER2-negative tumours (1·10 [1·02-1·19], p=0·011). INTERPRETATION: Increased TIL concentration predicted response to neoadjuvant chemotherapy in all molecular subtypes assessed, and was also associated with a survival benefit in HER2-positive breast cancer and TNBC. By contrast, increased TILs were an adverse prognostic factor for survival in luminal-HER2-negative breast cancer, suggesting a different biology of the immunological infiltrate in this subtype. Our data support the hypothesis that breast cancer is immunogenic and might be targetable by immune-modulating therapies. In light of the results in luminal breast cancer, further research investigating the interaction of the immune system with different types of endocrine therapy is warranted. FUNDING: Deutsche Krebshilfe and European Commission.

Outcome after neoadjuvant chemotherapy in estrogen receptor-positive and progesterone receptor-negative breast cancer patients: a pooled analysis of individual patient data from ten prospectively randomized controlled neoadjuvant trials.

PURPOSE: The estrogen receptor (ER) is involved in control of progesterone receptor (PgR) expression and lack of PgR may be also a surrogate of altered growth factor signaling. The aim of this study was therefore to investigate PgR expression as predictive factor for response to neoadjuvant therapy and long-term outcome. METHODS: Five thousand and six hundred and thirteen patients with primary breast cancer and positive ER expression from ten German neoadjuvant trials of anthracycline and taxane-based chemotherapy were included. Pathologic complete response (pCR), disease-free survival (DFS), distant disease-free survival (DDFS), overall survival (OS), and local recurrence-free survival (LRFS) were compared according to PgR expression. RESULTS: The lack of PgR expression (1172 patients) was associated with grade 3 (38.4 vs. 26.3%; p < 0.001), nodal involvement (>cN2) (6.8% vs. 4.7%; p = 0.004), and HER2 positivity (36.2 vs. 22.3%; p < 0.001). pCR rates of PgR-negative tumors were higher in the entire cohort (13.8 vs. 7.5%; p < 0.001) and in the HER2-negative subgroup (11.2 vs. 5.8%; p < 0.001). In multivariable logistic regression, PgR negativity was an independent predictive factor for pCR overall (OR 1.76; p < 0.001) and in the HER2-negative patients (OR 1.99; p < 0.001). Patients with PgR-negative disease had significantly worse outcome (p < 0.001, respectively). Multivariable Cox regression analysis revealed that PgR was an independent prognostic factor for DFS, OS, DDFS, and LRFS. CONCLUSION: ER-positive/PgR-negative breast carcinomas are associated with higher response but also worse long-term outcome after neoadjuvant therapy. PgR negativity is an independent predictive factor for pCR after neoadjuvant chemotherapy in ER-positive HER2-negative breast cancer.

Specific microRNA signatures in exosomes of triple-negative and HER2-positive breast cancer patients undergoing neoadjuvant therapy within the GeparSixto trial.

BACKGROUND: The focus of this study is to identify particular microRNA (miRNA) signatures in exosomes derived from plasma of 435 human epidermal growth factor receptor 2 (HER2)-positive and triple-negative (TN) subtypes of breast cancer (BC). METHODS: First, miRNA expression profiles were determined in exosomes derived from the plasma of 15 TNBC patients before neoadjuvant therapy using a quantitative TaqMan real-time PCR-based microRNA array card containing 384 different miRNAs. Forty-five miRNAs associated with different clinical parameters were then selected and mounted on microRNA array cards that served for the quantification of exosomal miRNAs in 435 BC patients before therapy and 20 healthy women. Confocal microscopy, Western blot, and ELISA were used for exosome characterization. RESULTS: Quantification of 45 exosomal miRNAs showed that compared with healthy women, 10 miRNAs in the entire cohort of BC patients, 13 in the subgroup of 211 HER2-positive BC, and 17 in the subgroup of 224 TNBC were significantly deregulated. Plasma levels of 18 exosomal miRNAs differed between HER2-positive and TNBC subtypes, and 9 miRNAs of them also differed from healthy women. Exosomal miRNAs were significantly associated with the clinicopathological and risk factors. In uni- and multivariate models, miR-155 (p = 0.002, p = 0.003, respectively) and miR-301 (p = 0.002, p = 0.001, respectively) best predicted pathological complete response (pCR). CONCLUSION: Our findings show a network of deregulated exosomal miRNAs with specific expression patterns in exosomes of HER2-positive and TNBC patients that are also associated with clinicopathological parameters and pCR within each BC subtype.

Validation of psychomotor tasks by Simbionix LAP Mentor simulator and identifying the target group.

BACKGROUND: This study addresses target group reliability and task validity for training on a laparoscopic simulator. MATERIAL AND METHODS: Data were collected on 64 participants prospectively at the Department of OB/GYN, University Hospitals Schleswig-Holstein, Campus Kiel. The Simbionix LAP Mentor for laparoscopic simulation was used to test trainees. Each participant received a questionnaire to clarify his/her medical position, surgical experience, and previous virtual reality (VR) experience, including video gaming experience. Pre- and post-tests were performed. Performances were analyzed for task completion and total time. RESULTS: All participants revealed a significant improvement in the post-test compared with the pre-test (p < .005), independent of their previous level of experience. Regarding accomplishment of the assigned task, the experts revealed in the pre-test an advantage in tasks 1-4 and 6-8. The beginners revealed wide-ranging improvements in tasks 3, 5, 8, and 9 between the pre-test and the post-test compared with novices in laparoscopic surgery (residents), and a wide range of improvements relative to experts. VR experience and video gaming exposure revealed an advantage in the pre-test; however, participants without previous exposure were able to narrow the gap, revealing extensive improvements in the post-test. CONCLUSION: The trainer could be beneficial for medical students and surgical novices.

Alteration of STR profiles in ovarian carcinoma cells during primary culture.

PURPOSE: Cell authentication is a necessary procedure to avoid scientific data from cell culture experiments with cross-contamination or false classification. A genetic fingerprint pattern of a specimen by short tandem repeats (STR) is self-evident. Due to high amount of chromosomal rearrangements, known in epithelia ovary cancer cells and the instable STR pattern described in other tumour entities like leukaemia, this study explores the suitability of STR profiling for primary cultured epithelial ovary cancer cells. METHODS: STR profiles of epithelial ovary cancers of 16 patients were compared with corresponding blood and corresponding primary cell cultures. The primary cell cultures of epithelial ovary tumours were passaged up to 28 times. In between, cultures were cryo conserved and recultured again, two to five times per patient. RESULTS: In two cases, the STR pattern of tumour lost alleles (1/16 and 3/16) in comparison of corresponding STR-pattern from blood. In comparison to blood, cell culture of a third case, lost four alleles (4/16) accompanied with morphologic changes after 14th passage. It is equal after cryo conservation of the seventh passage from the same patient. The only changes in STR profiles we recognized are losses of alleles. Remaining STR markers allow authentication. CONCLUSIONS: Very likely, the allelic drop-outs beyond passage 14 assume complex genetic losses of heterozygosis resulting in changed growth behaviour of cells. All other STR-profiles of remaining 15 patients analysed in this study are stable over all passages and freeze-thaw processes. Thus, ovary cancer cell cultures in research should be authenticated by STR-profile in general.

Head-to-head comparison of the impact of Aurora A, Aurora B, Repp86, CDK1, CDK2 and Ki67 expression in two of the most relevant gynaecological tumor entities.

PURPOSE: The dysregulation of cell cycle kinases plays a crucial role in carcinogenesis and the expression of various kinases has been attributed to aggressive tumor growth and an unfavourable prognosis in oncological patients. We, therefore, aimed to evaluate the expression of Ki67 among five additional cell cycle kinases in a collective of mammary and ovarian tumor specimens and to find a correlation with clinicopathological parameters. METHODS: 76 mammary and 93 ovarian benign and malignant tumor samples were immunohistochemically stained and evaluated for the expression of Aurora A and B, Repp86, CDK1 and 2 (only breast specimens) and Ki67. The expression patterns of these cell cycle kinases were matched with retrospectively collected clinicopathological parameters. RESULTS: All examined cell cycle kinases accurately discriminated benign from malignant breast and ovarian tissues. In breast cancer, Aurora A and B-, Repp86-, CDK2- and Ki67-expression was inversely associated with ER expression. No correlation with the HER2-status was found in our collective. Importantly, we found a significant correlation between the expression of Aurora A and CDK1 and axillary lymph node metastasis in breast cancer. Furthermore, a shortened disease free survival (DFS) upon expression of Aurora B and CDK2 was shown in breast cancer patients. None of the cell cycle kinases was associated with predictive or prognostic factors in epithelial ovarian cancer. CONCLUSION: The prognostic value of the expression of Ki67 is overtrumped by alternative cell cycle kinases when it comes to prediction of axillary tumor spread and a shortened DFS, which might allow a further risk stratification in breast cancer patients.

Genetic variants in VEGF pathway genes in neoadjuvant breast cancer patients receiving bevacizumab: Results from the randomized phase III GeparQuinto study.

Studies assessing the effect of bevacizumab (BEV) on breast cancer (BC) outcome have shown different effects on progression-free and overall survival, suggesting that a subgroup of patients may benefit from this treatment. Unfortunately, no biomarkers exist to identify these patients. Here, we investigate whether single nucleotide polymorphisms (SNPs) in VEGF pathway genes correlate with pathological complete response (pCR) in the neoadjuvant GeparQuinto trial. HER2-negative patients were randomized into treatment arms receiving either BEV combined with standard chemotherapy or chemotherapy alone. In a pre-planned biomarker study, DNA was collected from 729 and 724 patients, respectively from both treatment arms, and genotyped for 125 SNPs. Logistic regression assessed interaction between individual SNPs and both treatment arms to predict pCR. Five SNPs may be associated with a better response to BEV, but none of them remained significant after correction for multiple testing. The two SNPs most strongly associated, rs833058 and rs699947, were located upstream of the VEGF-A promoter. Odds ratios for the homozygous common, heterozygous and homozygous rare rs833058 genotypes were 2.36 (95% CI, 1.49-3.75), 1.20 (95% CI, 0.88-1.64) and 0.61 (95% CI, 0.34-1.12). Notably, some SNPs in VEGF-A exhibited a more pronounced effect in the triple-negative subgroup. Several SNPs in VEGF-A may be associated with improved pCR when receiving BEV in the neoadjuvant setting. Although none of the observed effects survived correction for multiple testing, our observations are consistent with previous studies on BEV efficacy in BC. Further research is warranted to clarify the predictive value of these markers.

In vitro and in vivo toxicity of 5-FdU-alendronate, a novel cytotoxic bone-seeking duplex drug against bone metastasis.

BACKGROUND: Bone remains one of the most common anatomic sites for cancer metastases, and the limited therapeutic options aggravate cancer-related morbidity and mortality in multiple malignancies. The covalent conjugation of the amino-bisphosphonate alendronate (ale) with the antimetabolite 5-fluoro-2'-desoxyuridine (5-FdU) results in N(4)-(butyl-(4-hydroxy-4-phosphono)phosphate)-5-fluoro-2'-desoxyuridine (5-FdU-alendronat, 5-FdU-ale), an effective, novel bone-targeting duplex drug directed against skeletal cancer manifestations. METHODS: In vitro cytotoxicity of ale, 5-FdU or 5-FdU-ale was measured with Alamar Blue and MUH cell viability assays in 14 malignant melanoma, multiple myeloma, bone marrow-derived stromal cell and osteoblast-like cell lines. In vivo toxicity was evaluated using the chicken embryo assay and evaluation of nephrotoxicity and the systemic toxicity in Balb/c nude mice. The effect of 5-FdU-ale on osteoclast was evaluated with Balb/c nude mice in a metastatic breast cancer mouse model. RESULTS: A cell line-specific, dose-related cytotoxicity was observed for 5-FdU-ale in all cancer cell lines tested, which was significantly less toxic than 5-FdU alone when compared to the benign osteoblasts or stromal cells. The embryotoxicity of 5-FdU-ale was significantly less than that of the parental drugs alendronate or 5-FdU. 5-FdU-ale showed no signs of unwanted side effects, weight loss or nephrotoxicity in mice. In a bone metastasis mouse model, 5-FdU-ale reduced the number of tumor-associated osteoclasts. CONCLUSION: The coupling of an amino-bisphosphonate with an antimetabolite via an N-alkyl-bonding offers a new strategy for the preparation of amino-bisphosphonates conjugates with a cancer cell-specific, efficacious cytotoxic bone-targeting potential along with a reduced systemic toxicity. The innovative duplex drug 5-FdU-ale therefore warrants further clinical investigation.

The incidence of complications by hysterectomy for benign disease in correlation to an assumed preoperative score.

BACKGROUND: Hysterectomies are preformed abdominally, vaginally, laparoscopic or with robotic assistance. When choosing the route of hysterectomy, the physician should take into consideration the safest and most cost-effective route to fulfill all needs of the patient. METHODS: Data were collected retrospectively from 953 patients who underwent hysterectomy between 2002 and 2010 for benign indications at UKSH, Germany. Preoperative risk scores were assigned to patients. The data were statistically evaluated to investigate relationship between the occurrence of the complication and the preoperative score at the time of the hysterectomy. For the preoperative score, patients who had undergone a previous laparoscopy were assigned 1 point; those who had undergone a previous Pfannenstiel laparotomy were assigned 2 points; those who had undergone 1 cesarean delivery were assigned 3 points; those who had undergone 2 cesarean deliveries were assigned 4 points; those who had undergone 3 cesarean deliveries were assigned 5 points; and those with no previous operations were assigned 0 points. The preoperative score was recorded for 785 patients. RESULTS: Of the 785 women with complete data, the mean preoperative score was 1.09 ± 1.51 for AH, 0.75 ± 0.96 for VH, 1.04 ± 1.30 for LSH, 1.0 ± 1.40 for LAVH, and 1.38 ± 1.52 for TLH. The prevalent scores in the VH were 0 and 1, the LASH and TLH showed a prevalence over VH in the preoperative scores 3 and 4 and AH showed a prevalence over the other methods in the preoperative score 3-8. Intraoperative complications were recorded in 28 of 953 (2.9 %) cases: 10 (35.7 %) cases of VH; 13 (46.4 %) cases of AH; 3 (10.7 %) cases of LSH; 1 (3.6 %) case of LAVH; and 1 (3.6 %) case of TLH. The intraoperative complications appeared to be more frequently with heavier uterine weight showing a significant statically correlation (P < 0,001). Major postoperative complications occurred in 17 of 953 (1.8 %) cases. Minor postoperative complications were recorded in 56 of 953 (5.9 %) hysterectomies. Operation duration, hospital stay and hemoglobin decline correlated significantly with preoperative score (P < 0.001). CONCLUSION: The suggested preoperative score is apparently successful in screening out the high-risk patients, despite the low incidence of intra and postoperative complications. The usefulness of the preoperative scoring system is worthy of further development and evaluation. The AH was favored as the 'fallback option' with high preoperative score.