On-treatment biopsies to predict response to neoadjuvant chemotherapy for breast cancer.

BACKGROUND: Patients with pathologic complete response (pCR) to neoadjuvant chemotherapy for invasive breast cancer (BC) have better outcomes, potentially warranting less extensive surgical and systemic treatments. Early prediction of treatment response could aid in adapting therapies. METHODS: On-treatment biopsies from 297 patients with invasive BC in three randomized, prospective neoadjuvant trials were assessed (GeparQuattro, GeparQuinto, GeparSixto). BC quantity, tumor-infiltrating lymphocytes (TILs), and the proliferation marker Ki-67 were compared to pre-treatment samples. The study investigated the correlation between residual cancer, changes in Ki-67 and TILs, and their impact on pathologic complete response (pCR) and disease-free survival (DFS). RESULTS: Among the 297 samples, 138 (46%) were hormone receptor-positive (HR+)/human epidermal growth factor 2-negative (HER2-), 87 (29%) were triple-negative (TNBC), and 72 (24%) were HER2+. Invasive tumor cells were found in 70% of on-treatment biopsies, with varying rates across subtypes (HR+/HER2-: 84%, TNBC: 62%, HER2+: 51%; p < 0.001). Patients with residual tumor on-treatment had an 8% pCR rate post-treatment (HR+/HER2-: 3%, TNBC: 19%, HER2+: 11%), while those without any invasive tumor had a 50% pCR rate (HR+/HER2-: 27%; TNBC: 48%, HER2+: 66%). Sensitivity for predicting residual disease was 0.81, with positive and negative predictive values of 0.92 and 0.50, respectively. Increasing TILs from baseline to on-treatment biopsy (if residual tumor was present) were linked to higher pCR likelihood in the overall cohort (OR 1.034, 95% CI 1.013-1.056 per % increase; p = 0.001) and with a longer DFS in TNBC (HR 0.980, 95% CI 0.963-0.997 per % increase; p = 0.026). Persisting or increased Ki-67 was associated with with lower pCR probability in the overall cohort (OR 0.957, 95% CI 0.928-0.986; p = 0.004) and shorter DFS in TNBC (HR 1.023, 95% CI 1.001-1.047; p = 0.04). CONCLUSION: On-treatment biopsies can predict patients unlikely to achieve pCR post-therapy. This could facilitate therapy adjustments for TNBC or HER2 + BC. They also might offer insights into therapy resistance mechanisms. Future research should explore whether standardized or expanded sampling enhances the accuracy of on-treatment biopsy procedures. Trial registration GeparQuattro (EudraCT 2005-001546-17), GeparQuinto (EudraCT 2006-005834-19) and GeparSixto (EudraCT 2011-000553-23).

The impact of physical activity on progression-free and overall survival in metastatic breast cancer based on molecular subtype.

BACKGROUND: Although adequate physical activity has been shown to be beneficial in early breast cancer, evidence in metastatic breast cancer is sparse and contradictory, which could be related to distinct effects of physical activity on the different molecular cancer subtypes. Therefore, we here evaluated the effect of physical activity on progression-free and overall survival (PFS, OS) in metastatic breast cancer, specifically looking at molecular subtypes. METHODS: International Physical Activity Questionnaire (IPAQ) questionnaires, filled out by patients enrolled in the prospective PRAEGNANT registry (NCT02338167; n = 1,270) were used to calculate metabolic equivalent task (MET) minutes, which were subsequently categorized into low (n = 138), moderate (n = 995) or high IPAQ categories (n = 137). Cox regression analyses were used to evaluate the impact of IPAQ categories and its interaction with molecular subtypes on PFS and OS. RESULTS: Patient and tumor characteristics were equally distributed across IPAQ categories. HER2pos, HRpos and TNBC were present in 23.1%, 65.7% and 11.2% of patients, respectively. IPAQ scores did not have an impact on PFS and OS in addition to established prognostic factors, either overall or in particular molecular subtypes (PFS: p = 0.33 and OS: p = 0.08, likelihood ratio test). Exploratory analyses showed higher overall survival rates for high IPAQ categories compared to low/moderate IPAQ categories in luminal B-like breast cancer. CONCLUSIONS: Self-reported physical activity using the IPAQ questionnaire did not significantly affect PFS or OS in patients suffering from metastatic breast cancer. Nevertheless, some hypothesis-generating differences between molecular subtypes could be observed, which may be interesting to evaluate further.

Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

Tucatinib, Trastuzumab, and Capecitabine for HER2-Positive Metastatic Breast Cancer.

BACKGROUND: Patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer who have disease progression after therapy with multiple HER2-targeted agents have limited treatment options. Tucatinib is an investigational, oral, highly selective inhibitor of the HER2 tyrosine kinase. METHODS: We randomly assigned patients with HER2-positive metastatic breast cancer previously treated with trastuzumab, pertuzumab, and trastuzumab emtansine, who had or did not have brain metastases, to receive either tucatinib or placebo, in combination with trastuzumab and capecitabine. The primary end point was progression-free survival among the first 480 patients who underwent randomization. Secondary end points, assessed in the total population (612 patients), included overall survival, progression-free survival among patients with brain metastases, confirmed objective response rate, and safety. RESULTS: Progression-free survival at 1 year was 33.1% in the tucatinib-combination group and 12.3% in the placebo-combination group (hazard ratio for disease progression or death, 0.54; 95% confidence interval [CI], 0.42 to 0.71; P<0.001), and the median duration of progression-free survival was 7.8 months and 5.6 months, respectively. Overall survival at 2 years was 44.9% in the tucatinib-combination group and 26.6% in the placebo-combination group (hazard ratio for death, 0.66; 95% CI, 0.50 to 0.88; P = 0.005), and the median overall survival was 21.9 months and 17.4 months, respectively. Among the patients with brain metastases, progression-free survival at 1 year was 24.9% in the tucatinib-combination group and 0% in the placebo-combination group (hazard ratio, 0.48; 95% CI, 0.34 to 0.69; P<0.001), and the median progression-free survival was 7.6 months and 5.4 months, respectively. Common adverse events in the tucatinib group included diarrhea, palmar-plantar erythrodysesthesia syndrome, nausea, fatigue, and vomiting. Diarrhea and elevated aminotransferase levels of grade 3 or higher were more common in the tucatinib-combination group than in the placebo-combination group. CONCLUSIONS: In heavily pretreated patients with HER2-positive metastatic breast cancer, including those with brain metastases, adding tucatinib to trastuzumab and capecitabine resulted in better progression-free survival and overall survival outcomes than adding placebo; the risks of diarrhea and elevated aminotransferase levels were higher with tucatinib. (Funded by Seattle Genetics; HER2CLIMB ClinicalTrials.gov number, NCT02614794.).

Return of individual genomic research results within the PRAEGNANT multicenter registry study.

PURPOSE: The PRAEGNANT study is a registry study for metastatic breast cancer patients, focusing on biomarker detection. Recently, within this study, genetic alterations in 37 breast cancer predisposition genes were analyzed and genetic findings were detected for 396 participants. The aim of this project was to return genetic results to the physicians and to analyze actions taken (e.g., disclosure of results to patients, validation of results, clinical impact, and impact on the patient's quality of life) using a questionnaire. METHODS: 235 questionnaires were sent out to the study centers, with each questionnaire representing one patient with a genetic finding. The questionnaire consisted of twelve questions in the German language, referring to the disclosure of results, validation of test results, and their impact on treatment decisions and on the patient's quality of life. RESULTS: 135 (57.5%) questionnaires were completed. Of these, 46 (34.1%) stated that results were returned to the patients. In 80.0% (N = 36) of cases where results were returned, the patient had not been aware of the finding previously. For 27 patients (64.3%), genetic findings had not been validated beforehand. All validation procedures (N = 15) were covered by the patients' health insurance. For 11 (25.0%) patients, physicians reported that the research results influenced current or future decision-making on treatment, and for 37.8% (N = 17) the results influenced whether family members will be genetically tested. CONCLUSION: This study provides novel insights into the return of research results and into clinical and personal benefits of disclosure of genetic findings within a German registry.

The role of the desmosomal protein desmocollin 2 in tumour progression in triple negative breast cancer patients.

BACKGROUND: The disruption of epithelial features represents a critical step during breast cancer spread. In this context, the dysregulation of desmosomal proteins has been associated with malignant progression and metastasis formation. Curiously, both tumour suppressive and pro-metastatic roles have been attributed to desmosomal structures in different cancer entities. In the present study, we describe the pro-metastatic role of the desmosomal protein desmocollin 2 (DSC2) in breast cancer. METHODS: We analysed the prognostic role of DSC2 at mRNA and protein level using microarray data, western blot analysis and immunohistochemistry. Functional consequences of DSC2 overexpression and DSC2 knock down were investigated in the triple negative breast cancer (TNBC) cell line MDA-MB-231 and its brain-seeking subline MDA-MB-231-BR, respectively in vitro and in vivo. RESULTS: We found a significantly higher DSC2 expression in the more aggressive molecular subtypes HER2-positive and TNBC than in luminal breast cancers, as well as a significant correlation between increased DSC2 expression and a shorter disease-free-also in multivariate analysis-and overall survival. Additionally, a significant association between DSC2 expression in the primary tumour and an increased frequency of cerebral and lung metastasis could be observed. In vitro, ectopic DSC2 expression or DSC2 down-regulation in MDA-MB-231 and MDA-MB-231-BR led to a significant tumour cell aggregation increase and decrease, respectively. Furthermore, tumour cells displaying higher DSC2 levels showed increased chemoresistance in 3D structures, but not 2D monolayer structures, suggesting the importance of cell aggregation as a means for reduced drug diffusion. In an in vivo brain dissemination xenograft mouse model, reduced expression of DSC2 in the brain-seeking TNBC cells led to a decreased amount of circulating tumour cells/clusters and, in turn, to fewer and smaller brain metastatic lesions. CONCLUSION: We conclude that high DSC2 expression in primary TNBC is associated with a poorer prognosis, firstly by increasing tumour cell aggregation, secondly by reducing the diffusion and effectiveness of chemotherapeutic agents, and, lastly, by promoting the circulation and survival of tumour cell clusters, each of which facilitates distant organ colonisation.

New treatment strategies for human epidermal growth factor receptor 2-positive breast cancer in 2023.

PURPOSE OF REVIEW: Taxanes in combination with trastuzumab and pertuzumab are the established first-line standard in the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. In the last years, several new HER2-targeted therapies, including antibody-drug conjugates (ADCs) and tyrosine kinase inhibitors, have been approved for therapy after trastuzumab or dual blockade. In this review, the current treatment algorithms are discussed, including these new treatment options. RECENT FINDINGS: The ADC T-DM1 was the established second-line standard based on the results of the EMILIA trial. Recently, the DESTINY-Breast03 trial compared T-DM1 with the new ADC trastuzumab deruxtecan (T-DXd) in patients with disease progression after treatment with taxanes and trastuzumab. T-DXd was associated with an improved progression-free survival and a trend toward improved overall survival, establishing T-DXd as a new second-line standard. The HER2CLIMB trial demonstrated a significant progression-free survival and overall survival benefit for the tyrosine kinase inhibitor tucatinib in combination with trastuzumab and capecitabine after T-DM1 and trastuzumab/pertuzumab. This benefit was also observed in patients with active brain metastases defining this combination as the preferred second or third-line option in these patients. SUMMARY: New treatment strategies in HER2-positive metastatic breast cancer have substantially improved the clinical outcome of these patients, including those with active brain metastases.

Major influencing factors on routine implementation of shared decision-making in cancer care: qualitative process evaluation of a stepped-wedge cluster randomized trial.

BACKGROUND: Shared decision-making (SDM) is highly relevant in oncology but rarely implemented in routine care. In a stepped-wedge cluster randomized implementation trial, the outcome evaluation of a theoretically and empirically based multi-component SDM implementation program did not show a statistically significant effect on patient-reported SDM uptake. Within this SDM implementation trial, a thorough a priori planned process evaluation was conducted. Thus, the aim of this study was to investigate factors influencing SDM implementation in the context of a multi-component SDM implementation program. METHODS: We conducted qualitative process evaluation of a stepped-wedge SDM implementation trial. Qualitative data included interviews with nurses and physicians of participating departments, field notes by the study team, and meeting minutes. Data were analyzed via deductive and inductive qualitative content analysis on basis of the Consolidated Framework for Implementation Research (CFIR). RESULTS: Transcripts of 107 interviews with 126 nurses and physicians, 304 pages of field note documentation, and 125 pages of meeting minutes were analyzed. Major factors influencing SDM implementation were found for all domains of the CFIR: a) four regarding characteristics of the individuals involved (e.g., perceived personal relevance, individual motivation to change), b) eleven regarding the inner setting (e.g., leadership engagement, networks and communication, available resources, compatibility with clinical practice), c) two regarding the outer setting (e.g., culture of health care delivery), d) eight regarding characteristics of the intervention (e.g., relative advantage, adaptability), and e) three regarding the implementation process (e.g., integration into existing structures). Furthermore, we found strong interrelations between several of the influencing factors within and between domains. CONCLUSIONS: This comprehensive process evaluation complements the outcome evaluation of the SDM implementation trial and adds to its interpretation. The identified influencing factors can be used for planning, conducting, and evaluating SDM implementation in the future. TRIAL REGISTRATION: clinicaltrials.gov, NCT03393351, registered 8 January 2018, https://clinicaltrials.gov/ct2/show/NCT03393351.

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.

Circulating Cellular Communication Network Factor 1 Protein as a Sensitive Liquid Biopsy Marker for Early Detection of Breast Cancer.

BACKGROUND: Despite recent progress in liquid biopsy technologies, early blood-based detection of breast cancer is still a challenge. METHODS: We analyzed secretion of the protein cellular communication network factor 1 (CCN1, formerly cysteine-rich angiogenic inducer 61) in breast cancer cell lines by an enzyme-linked immunosorbent assay (ELISA). Soluble CCN1 in the plasma (2.5 µL) of 544 patients with breast cancer and 427 healthy controls was analyzed by ELISA. The breast cancer samples were acquired at the time of primary diagnosis prior to neoadjuvant therapy or surgery. A classifier was established on a training cohort of patients with breast cancer and age-adapted healthy controls and further validated on an independent cohort comprising breast cancer patients and healthy controls. Samples from patients with benign breast diseases were investigated as additional controls. Samples from patients with acute heart diseases (n = 127) were investigated as noncancer controls. The diagnostic accuracy was determined by receiver operating characteristic using the parameters area under the curve, sensitivity, and specificity. RESULTS: CCN1 was frequently secreted by breast cancer cell lines into the extracellular space. Subsequent analysis of clinical blood samples from patients with breast cancer and age-adjusted healthy controls revealed an overall specificity of 99.0% and sensitivity of 80.0% for cancer detection. Remarkably, 81.5% of small T1 cancers were already CCN1-positive, while CCN1 concentrations in patients with benign breast lesions were below the threshold for breast cancer detection. CONCLUSIONS: Circulating CCN1 is a potentially novel blood biomarker for the detection of breast cancer at the earliest invasive stage.

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.

Myocardial injury detected by T1 and T2 mapping on CMR predicts subsequent cancer therapy-related cardiac dysfunction in patients with breast cancer treated by epirubicin-based chemotherapy or left-sided RT.

OBJECTIVES: Cancer therapy-related cardiac dysfunction (CTRCD) is a relevant clinical problem and needs early prediction. This study aimed to analyze myocardial injury using serial laboratory and cardiac magnetic resonance imaging (CMR) parameters after epirubicin-based chemotherapy compared with left-sided radiotherapy and to study their value for early prediction of CTRCD. METHODS: Sixty-six consecutive women (53 ± 13 years) including n = 39 with epirubicin-based chemotherapy and n = 27 with left-sided radiotherapy were prospectively studied by 3 T CMR including left ventricular (LV) mass and volumes for ejection fraction (LVEF), as well as feature-tracking with global longitudinal strain (GLS) and T1/T2 mapping. CMR was performed at baseline, at therapy completion (follow-up 1, FU1), and after 13 ± 2 months (FU2). CTRCD was defined as LVEF decline of at least 10% to < 55% or a > 15% GLS change at FU2. RESULTS: T1 and T2 increased at FU1 after epirubicin-based chemotherapy, but not after left-sided radiotherapy. CTRCD occurred in 20% of patients after epirubicin-based chemotherapy and in 4% after left-sided radiotherapy. T1 at FU1 was the best single parameter to predict CTRCD with an area under the curve (AUC) of 0.712 (CI 0.587-0.816, p = 0.005) with excellent sensitivity (100%, 66-100%), but low specificity (44%, 31-58%). Combined use of increased T1 and LVEF ≤ 60% at FU1 improved AUC to 0.810 (0.695-0.896) resulting in good sensitivity (78%, 44-95%) and specificity (84%, 72-92%). CONCLUSION: Only epirubicin-based chemotherapy, but not left-sided radiotherapy, resulted in increased T1/T2 myocardial relaxation times as a marker of myocardial injury. Combined use of CMR parameters may allow an early prediction of subsequent CTCRD. KEY POINTS: • Myocardial T1 and T2 relaxation times increased at FU1 after epirubicin-based chemotherapy, but not after left-sided radiotherapy. • Cancer therapy-related cardiac dysfunction (CTRCD) occurred in 20% of patients after epirubicin-based chemotherapy and in 4% after left-sided radiotherapy. • Combined use of increased T1 and reduced LVEF had an AUC of 0.810 (0.695-0.896) to predict CTRCD with good sensitivity (78%, 44-95%) and specificity (84%, 72-92%).

Insights into the Steps of Breast Cancer-Brain Metastases Development: Tumor Cell Interactions with the Blood-Brain Barrier.

Brain metastases (BM) represent a growing problem for breast cancer (BC) patients. Recent studies have demonstrated a strong impact of the BC molecular subtype on the incidence of BM development. This study explores the interaction between BC cells of different molecular subtypes and the blood-brain barrier (BBB). We compared the ability of BC cells of different molecular subtypes to overcome several steps (adhesion to the brain endothelium, disruption of the BBB, and invasion through the endothelial layer) during cerebral metastases formation, in vitro as well as in vivo. Further, the impact of these cells on the BBB was deciphered at the molecular level by transcriptome analysis of the triple-negative (TNBC) cells themselves as well as of hBMECs after cocultivation with BC cell secretomes. Compared to luminal BC cells, TNBC cells have a greater ability to influence the BBB in vitro and consequently develop BM in vivo. The brain-seeking subline and parental TNBC cells behaved similarly in terms of adhesion, whereas the first showed a stronger impact on the brain endothelium integrity and increased invasive ability. The comparative transcriptome revealed potential brain-metastatic-specific key regulators involved in the aforementioned processes, e.g., the angiogenesis-related factors TNXIP and CXCL1. In addition, the transcriptomes of the two TNBC cell lines strongly differed in certain angiogenesis-associated factors and in several genes related to cell migration and invasion. Based on the present study, we hypothesize that the tumor cell's ability to disrupt the BBB via angiogenesis activation, together with increased cellular motility, is required for BC cells to overcome the BBB and develop brain metastases.

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 .

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis.

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

Gemcitabine as adjuvant chemotherapy in patients with high-risk early breast cancer-results from the randomized phase III SUCCESS-A trial.

BACKGROUND: When chemotherapy is indicated in patients with early breast cancer, regimens that contain anthracyclines and taxanes are established standard treatments. Gemcitabine has shown promising effects on the response and prognosis in patients with metastatic breast cancer. The SUCCESS-A trial (NCT02181101) examined the addition of gemcitabine to a standard chemotherapy regimen in high-risk early breast cancer patients. METHODS: A total of 3754 patients with at least one of the following characteristics were randomly assigned to one of the two treatment arms: nodal positivity, tumor grade 3, age ≤ 35 years, tumor larger than 2 cm, or negative hormone receptor status. The treatment arms received either three cycles of 5-fluorouracil, epirubicin, and cyclophosphamide, followed by three cycles of docetaxel (FEC → Doc); or three cycles of FEC followed by three cycles of docetaxel and gemcitabine (FEC → Doc/Gem). The primary study aim was disease-free survival (DFS), and the main secondary objectives were overall survival (OS) and safety. RESULTS: No differences were observed in the 5-year DFS or OS between FEC → Doc and FEC → Doc/Gem. The hazard ratio was 0.93 (95% CI, 0.78 to 1.12; P = 0.47) for DFS and 0.94 (95% CI, 0.74 to 1.19; P = 0.60) for OS. For patients treated with FEC → Doc and FEC → Doc/Gem, the 5-year probabilities of DFS were 86.6% and 87.2%, and the 5-year probabilities of OS were 92.8% and 92.5%, respectively. CONCLUSION: Adding gemcitabine to a standard chemotherapy does not improve the outcomes in patients with high-risk early breast cancer and should therefore not be included in the adjuvant treatment setting. TRIAL REGISTRATION: Clinicaltrials.gov NCT02181101 and EU Clinical Trials Register EudraCT 2005-000490-21. Registered September 2005.

Clinical relevance of H-RAS, K-RAS, and N-RAS mRNA expression in primary breast cancer patients.

PURPOSE: The RAS family comprises three proto-oncogenes (H-RAS, K-RAS, and N-RAS) and is among the most widely studied of oncogenes. The present study aimed at investigating the clinical relevance of mRNA levels of the three isoforms in a large group of breast cancer patients with a long-term follow-up. METHODS: 198 previously untreated patients were enrolled in the study. mRNA levels of K-RAS, H-RAS, and N-RAS were measured using microarray (Affymetrix HG-U133A). RESULTS: Elevated H-RAS levels were found significantly more frequently in patients with larger (p = 0.021) and ER-positive tumors (p = 0.048), while elevated K-RAS levels were associated with nodal positivity (p = 0.001) and HER2-positivity (p = 0.010). Patients with high N-RAS mRNA levels were more likely to be diagnosed with triple-negativity (p < 0.001) and higher grading (p = 0.001). Patients with high K-RAS levels were more likely to show an elevated H-RAS (p = 0.003). After a median follow-up of 183 months, patients with high N-RAS expression had significantly reduced overall survival (OS) compared with patients with low N-RAS (mean: 146.9 vs. 211.0 months; median 169.3 vs. not reached; p = 0.009). In patients with non-metastatic disease at the time of tissue sampling, mean disease-free survival (DFS) was 150.1 months for patients with high N-RAS versus 227.7 months with low N-RAS; median DFS was not reached (p = 0.004). The expression of H-RAS and K-RAS was not associated with DFS/OS. In the multivariable analysis, distant metastasis, HER2 positivity, and elevated N-RAS mRNA levels independently predicted reduced OS, while nodal status, HER2 status, and N-RAS predicted reduced DFS. CONCLUSIONS: Elevated N-RAS mRNA levels predict impaired clinical outcome; hypothetically, further exploration of the RAS signaling pathway might enable identifying potential targeted treatment strategies. The association between high N-RAS levels and the most aggressive among breast cancer subtypes, the triple-negative phenotype, for which targeted approaches are still lacking, underlines the need to further investigate the RAS family.

Heregulin (HRG) assessment for clinical trial eligibility testing in a molecular registry (PRAEGNANT) in Germany.

BACKGROUND: Eligibility criteria are a critical part of clinical trials, as they define the patient population under investigation. Besides certain patient characteristics, clinical trials often include biomarker testing for eligibility. However, patient-identification mostly relies on the trial site itself and is often a time-consuming procedure, which could result in missing out on potentially eligible patients. Pre-selection of those patients using a registry could facilitate the process of eligibility testing and increase the number of identified patients. One aim with the PRAEGNANT registry (NCT02338167) is to identify patients for therapies based on clinical and molecular data. Here, we report eligibility testing for the SHERBOC trial using the German PRAEGNANT registry. METHODS: Heregulin (HRG) has been reported to identify patients with better responses to therapy with the anti-HER3 monoclonal antibody seribantumab (MM-121). The SHERBOC trial investigated adding seribantumab (MM-121) to standard therapy in patients with advanced HER2-negative, hormone receptor-positive (HR-positive) breast cancer and HRG overexpression. The PRAEGNANT registry was used for identification and tumor testing, helping to link potential HRG positive patients to the trial. Patients enrolled in PRAEGNANT have invasive and metastatic or locally advanced, inoperable breast cancer. Patients eligible for SHERBOC were identified by using the registry. Study aims were to describe the HRG positivity rate, screening procedures, and patient characteristics associated with inclusion and exclusion criteria. RESULTS: Among 2769 unselected advanced breast cancer patients, 650 were HER2-negative, HR-positive and currently receiving first- or second-line treatment, thus potentially eligible for SHERBOC at the end of current treatment; 125 patients also met further clinical eligibility criteria (e.g. menopausal status, ECOG). In the first/second treatment lines, patients selected for SHERBOC based on further eligibility criteria had a more favorable prognosis than those not selected. HRG status was tested in 38 patients, 14 of whom (36.8%) proved to be HRG-positive. CONCLUSION: Using a real-world breast cancer registry allowed identification of potentially eligible patients for SHERBOC focusing on patients with HER3 overexpressing, HR-positive, HER2-negative metastatic breast cancer. This approach may provide insights into differences between patients eligible or non-eligible for clinical trials. TRIAL REGISTRATION: Clinicaltrials, NCT02338167 , Registered 14 January 2015 - retrospectively registered.

High homogeneity of MMR deficiency in ovarian cancer.

OBJECTIVE: Mismatch repair (MMR) deficiency and Bethesda panel microsatellite instability (MSI) are increasingly analyzed to identify tumors that might benefit from immune checkpoint inhibitors, but tumor heterogeneity is a potential obstacle for such analyses. In ovarian cancer, data on intratumoral heterogeneity of MMR deficiency/MSI are lacking. METHODS: N = 582 ovarian cancers were screened for MMR deficiency by immunohistochemistry (IHC) on a tissue microarray. 10 cases suspect for MMR deficiency were identified among 478 interpretable cancers and repeated IHC on large sections combined with polymerase chain reaction (PCR)-based MSI analysis validated MMR deficiency/MSI in 9 of these tumors. RESULTS: MMR deficiency/MSI was predominantly seen in endmetrioid cancers (8 of 35, 23%) and also in 1 of 358 serous carcinomas (0.3%), but was absent in 34 mucinous carcinomas, 23 clear cell carcinomas, 17 malignant mixed Mullerian tumors (carcinosarcomas), and 11 mixed carcinomas. MMR deficiency involed protein loss of PMS2/MLH1 in 6 cases and of MSH2 and/or MSH6 in 3 cases. 7 MMR deficient cancers were MSI-high (all endometrioid), one was MSI-low (endometrioid) and one cancer with unequivocal MMR protein loss exhibited microsatellite stability (serous). MLH1 promotor methylation was observed in 4 of 5 endometrioid cancers with MLH1 protein loss. Immunostaining of all available cancer-containing tissue blocks (n = 114) of tumors with confirmed MMR deficiency/MSI revealed uniform MMR status throughout the entire tumor mass. CONCLUSIONS: Our data show that MSI is present in a substantial proportion of endometrioid ovarian cancers but can also occur in other tumor subtypes. MMR deficiency/MSI typically involves the entire tumor mass, suggesting that MMR inactivation occurs early in tumorigenesis in a subset of ovarian cancers.