A multicentre, randomised, double-blind, phase II study to evaluate the tolerability of an induction dose escalation of everolimus in patients with metastatic breast cancer (DESIREE).

BACKGROUND: Stomatitis is one of the main reasons to discontinue everolimus in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC). To decrease stomatitis and subsequently early treatment discontinuations or dose reductions, the DESIREE trial investigated the use of a stepwise dose-escalation schedule of everolimus (EVE esc). PATIENTS AND METHODS: DESIREE is a phase II, multicentre, randomised, double-blind, placebo-controlled trial in patients with HR+/HER2- mBC and progression/relapse after nonsteroidal aromatase inhibitor treatment. Patients were randomised to EVE esc (2.5 mg/day, week 1; 5 mg/day, week 2; 7.5 mg/day, week 3; 10 mg/day, weeks 4-24) or everolimus 10 mg/day (EVE 10mg) for 24 weeks plus exemestane. The primary endpoint was the incidence of stomatitis episodes grade ≥2 within 12 weeks of treatment. The secondary endpoints included toxicity, relative total dose intensity (RTDI) and quality of life (QoL). RESULTS: A total of 160 patients were randomised and 156 started treatment (EVE esc: 80; EVE 10mg: 76). The median age of patients was 64 years (range 33-85), 56.3% patients in the EVE esc arm versus 42.1% in the EVE 10mg arm had liver metastasis (P = 0.081) and 62.5% versus 51.3% received over one metastatic therapy line (P = 0.196). Within 12 weeks, the incidence of stomatitis episodes grade ≥2 was significantly lower in the EVE esc arm compared with the EVE 10mg arm (28.8% versus 46.1%; odds ratio 0.47, 95% confidence interval 0.24-0.92; P = 0.026). Toxicity was in line with the known safety profile without new safety concerns. The median RTDI was 91.1% in the EVE esc arm versus 80.0% in the EVE 10mg arm (P = 0.329). Discontinuation rate in the first 3 weeks was 6.3% versus 15.8%, respectively (P = 0.073). QoL was comparable between the two treatment arms. CONCLUSIONS: A dose-escalation schema of everolimus over 3 weeks can be successfully used to reduce the incidence of high-grade stomatitis in the first 12 weeks of treatment in patients with HR+/HER2- mBC. TRIAL REGISTRATION: ClinicalTrials.govNCT02387099; https://clinicaltrials.gov/ct2/show/NCT02387099.

The age-specific differences in histopathological tumor characteristics and TNM classification of breast carcinomas in Quality assured mamma diagnostic (QuaMaDi) program in the state of Schleswig-Holstein in Germany.

BACKGROUND: We explored the hypothesis that high-quality standards in diagnostic mammography can lead to an early diagnosis of breast cancers and identifies at risk populations outside screening programs. The histopathological features and distribution of the TNM classification were examined in relation to patient age in a large group of women with breast cancers participating in the Quality Assured Mamma Diagnostic (QuaMaDi) program of the state of Schleswig-Holstein. PATIENTS AND METHODS: Surgical pathological reports were studied for clinicopathological characteristics, receptor status, molecular subtype and tumor stage. The analysis was conducted by dividing the study population into three age groups: women under 50 years (pre-screening), 50-69 years (peri-screening) and over 70 years (post-screening). RESULTS: 7.111 biopsies and 2.887 resection specimens were included. Breast cancer was diagnosed in 4.241 (59.7%) cases, one fourth of them in women < 50 years. Elderly women (> 70 years) had more well-differentiated, estrogen receptor (ER)-positive and HER2-negative carcinomas, whereas younger women (< 50 years) tended to have more poorly differentiated, ER negative, and HER2-positive carcinomas. 47% of breast carcinoma were luminal B tumors and were most common regardless of age. 70.4% of resected specimen had pT1 stage. Nodal negative were 71.2%. CONCLUSION: In QuaMaDi breast cancer was diagnosed at an early and potentially curable stage of the disease due to high-quality standards in diagnostic mammography. In addition, regardless of age, an increased number of prognostically unfavorable molecular subtypes were detected. Thus, QuaMaDi helps to identify at risk populations. QuaMaDi significantly improves diagnostic mammography and complements mammography screening programs.

Association of RAD51 with homologous recombination deficiency (HRD) and clinical outcomes in untreated triple-negative breast cancer (TNBC): analysis of the GeparSixto randomized clinical trial.

BACKGROUND: Current genetic and genomic tests measuring homologous recombination deficiency (HRD) show limited predictive value. This study compares the performance of an immunohistology-based RAD51 test with genetic/genomic tests to identify patients with HRD primary triple-negative breast cancer (TNBC) and evaluates its accuracy to select patients sensitive to platinum-based neoadjuvant chemotherapy (NACT). PATIENTS AND METHODS: This is a retrospective, blinded, biomarker analysis from the GeparSixto randomized clinical trial. TNBC patients received neoadjuvant paclitaxel plus Myocet®-nonpegylated liposomal doxorubicin (PM) or PM plus carboplatin (PMCb), both arms including bevacizumab. Formalin-fixed paraffin-embedded (FFPE) tumor samples were laid on tissue microarrays. RAD51, BRCA1 and γH2AX were quantified using an immunofluorescence assay. The predictive value of RAD51 was assessed by regression models. Concordance analyses were carried out between RAD51 score and tumor BRCA (tBRCA) status or genomic HRD score (Myriad myChoice®). Associations with pathological complete response (pCR) and survival were studied. Functional HRD was predefined as a RAD51 score ≤10% (RAD51-low). RESULTS: Functional HRD by RAD51-low was evidenced in 81/133 tumors (61%). RAD51 identified 93% tBRCA-mutated tumors and 45% non-tBRCA mutant cases as functional HRD. The concordance between RAD51 and genomic HRD was 87% [95% confidence interval (CI) 79% to 93%]. In patients with RAD51-high tumors, pCR was similar between treatment arms [PMCb 31% versus PM 39%, odds ratio (OR) 0.71, 0.23-2.24, P = 0.56]. Patients with RAD51-low tumors benefited from PMCb (pCR 66% versus 33%, OR 3.96, 1.56-10.05, P = 0.004; interaction test P = 0.02). This benefit maintained statistical significance in the multivariate analysis. Carboplatin addition showed similar disease-free survival in the RAD51-high [hazard ratio (HR) 0.40, log-rank P = 0.11] and RAD51-low (0.45, P = 0.11) groups. CONCLUSIONS: The RAD51 test identifies tumors with functional HRD and is highly concordant with tBRCA mutation and genomic HRD. RAD51 independently predicts clinical benefit from adding Cb to NACT in TNBC. Our results support further development to incorporate RAD51 testing in clinical decision-making.

Reconstructing tumor history in breast cancer: signatures of mutational processes and response to neoadjuvant chemotherapy⋆.

BACKGROUND: Different endogenous and exogenous mutational processes act over the evolutionary history of a malignant tumor, driven by abnormal DNA editing, mutagens or age-related DNA alterations, among others, to generate the specific mutational landscape of each individual tumor. The signatures of these mutational processes can be identified in large genomic datasets. We investigated the hypothesis that genomic patterns of mutational signatures are associated with the clinical behavior of breast cancer, in particular chemotherapy response and survival, with a particular focus on therapy-resistant disease. PATIENTS AND METHODS: Whole exome sequencing was carried out in 405 pretherapeutic samples from the prospective neoadjuvant multicenter GeparSepto study. We analyzed 11 mutational signatures including biological processes such as APOBEC-mutagenesis, homologous recombination deficiency (HRD), mismatch repair deficiency and also age-related or tobacco-induced alterations. RESULTS: Different subgroups of breast carcinomas were defined mainly by differences in HRD-related and APOBEC-related mutational signatures and significant differences between hormone-receptor (HR)-negative and HR-positive tumors as well as correlations with age, Ki-67 and immunological parameters were observed. We could identify mutational processes that were linked to increased pathological complete response rates to neoadjuvant chemotherapy with high significance. In univariate analyses for HR-positive tumors signatures, S3 (HRD, P < 0.001) and S13 (APOBEC, P = 0.001) as well as exonic mutation rate (P = 0.002) were significantly correlated with increased pathological complete response rates. The signatures S3 (HRD, P = 0.006) and S4 (tobacco, P = 0.011) were prognostic for reduced disease-free survival of patients with chemotherapy-resistant tumors. CONCLUSION: The results of this investigation suggest that the clinical behavior of a tumor, in particular, response to neoadjuvant chemotherapy and disease-free survival of therapy-resistant tumors, could be predicted by the composition of mutational signatures as an indicator of the individual genomic history of a tumor. After additional validations, mutational signatures might be used to identify tumors with an increased response rate to neoadjuvant chemotherapy and to define therapy-resistant subgroups for future therapeutic interventions.

Tumor mutational burden and immune infiltration as independent predictors of response to neoadjuvant immune checkpoint inhibition in early TNBC in GeparNuevo.

BACKGROUND: The predictive value of tumor mutational burden (TMB), alone or in combination with an immune gene expression profile (GEP), for response to neoadjuvant therapy in early triple negative breast cancer (TNBC) is currently not known, either for immune checkpoint blockade (ICB) or conventional chemotherapy. PATIENTS AND METHODS: We obtained both whole exome sequencing and RNA-Seq data from pretreatment samples of 149 TNBC of the recent neoadjuvant ICB trial, GeparNuevo. In a predefined analysis, we assessed the predictive value of TMB and a previously developed immune GEP for pathological complete remission (pCR). RESULTS: Median TMB was 1.52 mut/Mb (range 0.02-7.65) and was significantly higher in patients with pCR (median 1.87 versus 1.39; P = 0.005). In multivariate analysis, odds ratios for pCR per mut/Mb were 2.06 [95% confidence intervals (CI) 1.33-3.20, P = 0.001] among all patients, 1.77 (95% CI 1.00-3.13, P = 0.049) in the durvalumab treatment arm, and 2.82 (95% CI 1.21-6.54, P = 0.016) in the placebo treatment arm, respectively. We also found that both continuous TMB and immune GEP (or tumor infiltrating lymphocytes) independently predicted pCR. When we stratified patients in groups based on the upper tertile of TMB and median GEP, we observed a pCR rate of 82% (95% CI 60% to 95%) in the group with both high TMB and GEP in contrast to only 28% (95% CI 16% to 43%) in the group with both low TMB and GEP. CONCLUSIONS: TMB and immune GEP add independent value for pCR prediction. Our results recommend further analysis of TMB in combination with immune parameters to individually tailor therapies in breast cancer.

Impact of Histopathological Factors, Patient History and Therapeutic Variables on Recurrence-free Survival after Ductal Carcinoma in Situ: 8-Year Follow-up and Questionnaire Survey.

Introduction: Ductal carcinoma in situ (DCIS) is a premalignant lesion of the glandular component of the breast and a precursor lesion of invasive breast cancer. In recent decades the incidence of DCIS has risen continuously, mainly because of more extensive screening and more advanced diagnostic procedures. There is an increasing need for evidence-based treatment guidelines which will protect patients as far as possible from recurrence or invasive cancer but also from overtreatment. This retrospective single-center clinical trial analyzed recurrence-free survival times, rates of invasive and non-invasive events, and the impact of patient history, histopathological variables and therapeutic factors on recurrence-free survival times. Material and Methods: A total of 200 patients who underwent surgery between 2000 and 2007 for pure DCIS were included in the study. As part of follow-up a questionnaire was sent to patients and their respective gynecologists. Results: In the follow-up period, 12.5 % (n = 25) of the 200 patients had recurrence (invasive or non-invasive event). Menopausal status, tumor grade and tumor size were significantly associated with recurrence. Low-grade DCIS was significantly more often hormone receptor-positive than high-grade DCIS. Patients who had postoperative radiotherapy significantly more often also received endocrine drug treatment. There was a significant association between younger patient age and drug treatment. The study found that in the investigated cohort, premenopausal women had a significantly shorter recurrence-free time compared to postmenopausal women. Conclusion: This paper summarizes the current literature on DCIS. There is a need for more prospective clinical trials to improve the prognosis of premenopausal women with large and hormone receptor-positive DCIS.

Regulation of beta2-microglobulin expression in different human cell lines by proinflammatory cytokines.

BACKGROUND: Proinflammatory monocytic cytokines such as interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and IL-6 have been incriminated in the pathogenesis of elevated beta2-microglobulin (beta2M) serum concentrations in patients undergoing haemodialysis with so-called bioincompatible dialyser membranes. However, neither the source of the elevated serum beta2M nor the precise role of monocytic cytokines in the expression of the beta2M gene have been elucidated conclusively. The aim of the current study was to evaluate whether monocytic cytokines, and in particular IL-6, are regulators of beta2M gene expression in human hepatoma cells, T-lymphocytes and monocytes. METHODS: HepG2 and HuH7 human hepatoma cells, Jurkat T-cells, monocytic MonoMac6 cells, primary human monocytes and synoviocytes were stimulated with IL-1beta, IL-6, interferon-alpha (IFN-alpha), IFN-gamma or conditioned media from lipopolysaccharide (LPS)-treated monocytes. Expression of beta2M mRNA was analysed by Northern blotting, beta2M protein synthesis was determined by metabolic labelling and immunoprecipitation, and beta2M secretion was measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: In all cell types tested, IFN-gamma and, to a lesser extent, IFN-alpha stimulated gene expression of beta2M resulting in an increased synthesis and secretion of beta2M protein. Neither IL-1beta and IL-6 nor supernatants from LPS-treated monocytes were capable of inducing beta2M gene expression, with the exception of a small increase in HuH7 hepatoma cells upon IL-1beta treatment. CONCLUSIONS: The present study provides evidence that interferons are important regulators of beta2M expression. It also shows that proinflammatory monocytic cytokines do not modulate directly the expression of beta2M in cells of hepatic, monocytic and T-lymphocytic origin. Whether they influence beta2M synthesis and secretion indirectly by modulating interferon synthesis needs to be elucidated.

Clostridium botulinum C3 exoenzyme stimulates GLUT4-mediated glucose transport, but not glycogen synthesis, in 3T3-L1 adipocytes--a potential role of rho?

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role of PI-3-kinase and small GTP-binding proteins has been proposed. In previous studies we, among many others, excluded a role for the ras/MAP kinase pathway in insulin-mediated glucose transport. In this study we examined a possible role of the small GTP-binding protein rho in this process. Pretreatment of 3T3-L1 adipocytes with botulinum C3 exoenzyme (C3), which is known to ADP-ribosylate and inactivate rho, potently stimulated glucose uptake to a level similar to insulin. Interestingly, glycogen synthesis was not affected by C3 treatment. Insulin stimulates glucose uptake by triggering the translocation of GLUT4, the insulin-sensitive glucose transporter isotype, from an intracellular compartment to the plasma membrane. Similarly, C3-induced glucose uptake was paralleled by GLUT4 translocation. These data point to an important and novel role of the target of C3 (likely rho) in the regulation of GLUT4-mediated glucose transport. Our data suggest that insulin might stimulate glucose uptake through inactivation of rho.

Protein kinase C activation inhibits receptor-evoked inositol trisphosphate formation and induction of cytosolic calcium oscillations by decreasing the affinity-state of the cholecystokinin receptor in pancreatic acinar cells.

Digital-imaging microscopy of Fura-2-loaded pancreatic acinar cells revealed that the C-terminal octapeptide of cholecystokinin (CCK8) dose-dependently recruited 94% of freshly isolated acinar cells in terms of receptor-evoked Ca2+ mobilization. Maximal and half-maximal cell-recruitment were reached with 0.1 nM and 16.8 pM CCK8, respectively. The upstroke of the dose-recruitment curve consisted of cells displaying oscillatory changes in free cytosolic Ca2+ concentration ([Ca2+]i). After having reached its maximum, the percentage oscillating cells dose-dependently decreased upon further increasing of the CCK8 concentration. Pretreatment of the acinar cells with 0.1 microM TPA caused a rightward shift of the dose-recruitment curve but did not change the maximal effect of CCK8 on the recruitment of oscillating cells. Half-maximal recruitment was obtained with 287 pM CCK8. This observation demonstrates that high levels of protein kinase C activation do not inhibit Ca2+ oscillations at a level downstream to receptor activation. Moreover, this observation demonstrates that protein kinase C-mediated inhibition of Ca2+ oscillations evoked by submaximal CCK8 concentrations occurs at the receptor level, converting it from a high-affinity state into a low-affinity state. This conclusion is supported by the observation that TPA completely inhibited the recruitment of acinar cells in response to the high-affinity receptor agonist JMV-180. The inhibitory action of TPA on CCK8-evoked cell-recruitment was paralleled by an inhibitory effect of the phorbol ester on the CCK8-evoked peak increase in average inositol trisphosphate concentration in a population of acinar cells. This observation indicates that low concentrations of CCK8 interact with the high-affinity CCK receptor to increase [Ca2+]i through the intermediation of inositol trisphosphate.

Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state.

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca2+ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokinin-octapeptide (CCK8) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca2+ concentration ([Ca2+]i,av) in a suspension of acinar cells. However, in the presence of 1.0 microM staurosporine the stimulatory effect of submaximal concentrations of CCK8 was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca2+]i,av in cells prestimulated with a submaximal concentration of CCK8. The data obtained with staurosporine indicate that CCK8-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca2+ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK8-induced Ca2+ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK8-evoked increase in [Ca2+]i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK8 at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca2+]i,av evoked by the CCK8 analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca2+]i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK8 concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca2+]i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca2+ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)