Transition-Metal-Mediated Chemo- and Stereoselective Total Synthesis of (-)-Galanthamine.

An asymmetric synthetic route to (-)-galanthamine (1), a pharmacologically active Amaryllidaceae alkaloid used for the symptomatic treatment of early onset Alzheimer's disease, was successfully established with very high levels of stereocontrol. The key to achieving high chemo- and stereo-selectivity in this approach was the use of transition-metal-mediated reactions, namely, enyne ring-closing metathesis, Heck coupling, and titanium-based asymmetric allylation.

Control of the Restriction Point by Rb and p21.

The Restriction Point was originally defined as the moment that cells commit to the cell cycle and was later suggested to coincide with hyperphosphorylation of the retinoblastoma protein (Rb). Current cell cycle models posit that cells exit mitosis into a pre-Restriction Point state, where they have low cyclin-dependent kinase (CDK) activity and hypophosphorylated Rb; passage through the Restriction Point then occurs in late G1. Recent single-cell studies have challenged the current paradigm, raising questions about the location of the Restriction Point and the notion that cells exit mitosis into a pre-Restriction Point state. Here, we use a variety of single-cell techniques to show that both noncancer and cancer cells bifurcate into two subpopulations after anaphase, marked by increasing vs. low CDK2 activity and hyper- vs. hypophosphorylation of Rb. Notably, subpopulations with hyper- and hypophosphorylated Rb are present within minutes after anaphase, delineating one subpopulation that never "uncrosses" the Restriction Point and continues cycling and another subpopulation that exits mitosis into an uncommitted pre-Restriction Point state. We further show that the CDK inhibitor p21 begins rising in G2 in mother cells whose daughters exit mitosis into the pre-Restriction Point, CDK2low state. Furthermore, degradation of p21 coincides with escape from the CDK2low state and passage through the Restriction Point. Together, these data support a model in which only a subset of cells returns to a pre-Restriction Point state after mitosis and where the Restriction Point is sensitive to not only mitogens, but also inherited DNA replication stress via p21.

Lactate concentration in breast cancer using advanced magnetic resonance spectroscopy.

BACKGROUND: Precision medicine in breast cancer demands markers sensitive to early treatment response. Aerobic glycolysis (AG) upregulates lactate dehydrogenase A (LDH-A) with elevated lactate production; however, existing approaches for lactate quantification are either invasive or impractical clinically. METHODS: Thirty female patients (age 39-78 years, 15 grade II and 15 grade III) with invasive ductal carcinoma were enrolled. Lactate concentration was quantified from freshly excised whole tumours with double quantum filtered (DQF) magnetic resonance spectroscopy (MRS), and Nottingham Prognostic Index (NPI), LDH-A and proliferative marker Ki-67 were assessed histologically. RESULTS: There was a significantly higher lactate concentration (t = 2.2224, p = 0.0349) in grade III (7.7 ± 2.9 mM) than in grade II (5.5 ± 2.4 mM). Lactate concentration was correlated with NPI (ρ = 0.3618, p = 0.0495), but not with Ki-67 (ρ = 0.3041, p = 0.1023) or tumour size (r = 0.1716, p = 0.3645). Lactate concentration was negatively correlated with LDH-A (ρ = -0.3734, p = 0.0421). CONCLUSION: Our results showed that lactate concentration in whole breast tumour from DQF MRS is sensitive to tumour grades and patient prognosis.

A map of protein dynamics during cell-cycle progression and cell-cycle exit.

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence.

Design and synthesis of a potent, highly selective, orally bioavailable, retinoic acid receptor alpha agonist.

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARß, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARß (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.

Tumor-infiltrating lymphocytes and breast cancer mortality in racially and ethnically diverse participants of the Northern California Breast Cancer Family Registry.

Stromal tumor-infiltrating lymphocyte (sTIL) enrichment in pretreatment breast tumors has been associated with superior response to neoadjuvant treatment and survival. In a population-based cohort, we studied sTIL-survival associations by race and ethnicity. We assessed associations of continuous sTIL scores and sTIL-enriched breast cancers (defined as percent lymphocytic infiltration of tumor stroma or cell nests at cutoffs of 30%, 50%, and 70%) with clinical and epidemiologic characteristics and conducted multivariable survival analyses. Although we identified no difference in sTIL score by race and ethnicity, higher continuous sTIL score was associated with lower breast cancer-specific mortality only among non-Hispanic White and Asian American but not African American and Hispanic women. This finding suggests that complex factors influence treatment response and survival, given that sTIL enrichment was not associated with a survival advantage among women from minoritized groups, who more often experience health disparities. Further study of patient selection for sTIL-guided treatment strategies is warranted.

SerpinB3, a new prognostic tool in breast cancer patients treated with neoadjuvant chemotherapy.

It is unclear which patients with breast cancer benefit from anthracycline-based neoadjuvant chemotherapy and whether taxanes increase survival. Hsp70 and serpinB3 inhibit a lysosomal cell death pathway induced in anthracycline and taxane treated cells, which may be critical for breast cancer cell survival. Thus we evaluated serpinB3 and Hsp70 as putative prognostic biomarkers in breast cancer patients treated with neoadjuvant chemotherapy. SerpinB3 and Hsp70 were measured by immunohistochemistry in residual breast tumours of patients without a complete pathological response [pCR] (n = 250), from a retrospective cohort of 296 patients treated with anthracycline-based chemotherapy with or without sequential docetaxel prior to surgical resection. SerpinB3 (P = 0.02) and Hsp70 (P = 0.008) positivity in residual tumour were associated with a poor pathological response and serpinB3 was an independent prognostic biomarker (HR 2.1 (95% CI 1.2-3.8), P = 0.02). Docetaxel significantly improved overall survival of breast cancer patients treated with neoadjuvant chemotherapy. Furthermore, serpinB3 positivity predicted poor survival in patients treated with anthracycline-based chemotherapy alone (P = 0.02), but those with serpinB3 negative tumours had as equally good survival as those also treated with docetaxel (P = 0.7). Survival was independent of serpinB3 expression in patients who received sequential docetaxel. The Nottingham prognostic index (NPI), calculated at surgical resection, predicted overall survival in these neoadjuvantly treated patients (P < 0.001) and serpinB3 status segregated patients with a moderate NPI into distinct prognostic subgroups. The use of clinical (NPI) and molecular (serpinB3) biomarkers measured at surgical resection to provide accurate prognostication in patients who do not achieve a pCR following neoadjuvant chemotherapy could facilitate optimal post-operative clinical management of these patients and is of significant clinical value. Furthermore, serpinB3 status in residual tumour is a biomarker of neoadjuvant docetaxel benefit in patients not achieving a pCR and use of serpinB3 molecular subtyping for adjuvant docetaxel treatment planning warrants further investigation.

Characterisation of tumour-infiltrating macrophages: impact on response and survival in patients receiving primary chemotherapy for breast cancer.

The role of the tumour microenvironment and complex cellular interactions has attracted interest in responses to primary chemotherapy. Of particular interest are tumour-infiltrating T cells and tumour-infiltrating macrophages (TIMs). We evaluated TIMs and their key activation markers in patients with breast cancer undergoing primary chemotherapy related to response and survival. One hundred and ninety nine patients with large or locally advanced breast cancers received primary chemotherapy. Clinical data, histopathological responses to chemotherapy and survival were examined related to infiltrating cells in tumour microenvironments: cluster of differentiation (CD)3 (pan T cell); CD4 (helper T cells); CD8 (cytotoxic T cells); CD25 (activated T cells); CD68, suppressor of cytokine signalling (SOCS)1, SOCS3 (macrophages); and CD11c and CD205 (dendritic). In tumours demonstrating better responses to chemotherapy, there were significantly fewer CD4(+) T-helper cells than a poorer response (p < 0.05). There were increased numbers of SOCS3 expressing macrophages (pro-inflammatory) in tumours with complete pathological responses compared with no response to chemotherapy (p < 0.05). There was no association between SOCS1 expressing macrophages (anti-inflammatory) and tumour response. Multivariate analysis revealed that factors indicating better survival were receiving anthracycline plus docetaxel (ExpB = 1.166; p = 0.006), better pathological chemotherapy response (ExpB = 0.309; p = 0.009) and a low macrophage SOCS1 expression (ExpB = 13.465; p = 0.044). This study highlights the heterogeneity of TIMs and provides further insight into complex interactions within tumours. The results emphasise the importance of characterising activation status of infiltrating macrophages and provides proof of principle for using macrophage SOCS protein expression as a survival predictor. The apparent impact of macrophage subsets on overall survival underlines the therapeutic potential of manipulating macrophage activation in cancer.

Profiling the expression of cytochrome P450 in breast cancer.

AIMS: The cytochrome P450s (P450) are key oxidative enzymes that metabolize many carcinogens and anticancer drugs. Thus, these enzymes influence tumour development, tumour response to therapy and are putative tumour biomarkers. The aim was to define the P450 expression profile in breast cancer and establish the significance of P450 expression in this tumour type. METHODS AND RESULTS: A tissue microarray containing 170 breast cancers of no special type was immunostained for a panel of 21 P450s. The highest percentage of strong immunopositivity in breast cancers was seen for CYP4X1 (50.8%), CYP2S1 (37.5%) and CYP2U1 (32.2%), while CYP2J (98.6%) and CYP3A43 (70.7%) were the P450s that most frequently displayed no immunoreactivity. CYP4V2 (P = 0.01), CYP4X1 (P = 0.01) and CYP4Z1 (P = 0.01) showed correlations with tumour grade. CYP1B1 (P = 0.001), CYP3A5 (P = 0.001) and CYP51 (P = 0.005) showed the most significant correlations with oestrogen receptor status. Correlations with survival were identified for CYP2S1 (P = 0.03), CYP3A4 (P = 0.025), CYP4V2 (P = 0.026) and CYP26A1 (P = 0.03), although none of these P450s was an independent marker of prognosis. CONCLUSIONS: This study has defined the expression profile of cytochrome P450s in breast cancer and may offer their potential application as biomarkers to aid decisions regarding optimal adjuvant hormonal therapy.

Anaplastic transformation in lung metastases of differentiated papillary thyroid carcinoma: an autopsy case report and review of the literature.

Anaplastic transformation of papillary thyroid carcinoma is a rare condition and is associated with an aggressive behavior. Most cases described in the literature have occurred in the thyroid gland or in the surrounding cervical lymph nodes. We report a case of an 83-year-old man who, 10 years after being treated for a conventional papillary thyroid carcinoma, presented with widespread metastases in his lungs. At autopsy, he was found to have extensive metastatic deposits in both lungs, which histologically and immunohistochemically were consistent with papillary thyroid carcinoma. An unusual finding was the presence of solid pleomorphic foci within tumor deposits, consistent with anaplastic transformation.

Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence.

Ki67 staining is widely used as a proliferation indicator in the clinic, despite poor understanding of this protein's function or dynamics. Here, we track Ki67 levels under endogenous control in single cells over time and find that Ki67 accumulation occurs only during S, G2, and M phases. Ki67 is degraded continuously in G1 and G0 phases, regardless of the cause of entry into G0/quiescence. Consequently, the level of Ki67 during G0 and G1 in individual cells is highly heterogeneous and depends on how long an individual cell has spent in G0. Thus, Ki67 is a graded rather than a binary marker both for cell-cycle progression and time since entry into quiescence.

Avoiding the danger that stop smoking services may exacerbate health inequalities: building equity into performance assessment.

BACKGROUND: The UK is the only developed country to have established a nation-wide stop smoking treatment service. Apart from addressing tobacco dependence, which is the leading preventable cause of ill health and premature death, smoking cessation has been identified by the UK department of health as a service priority for reducing gaps in health between disadvantaged groups and the country as a whole. However smoking cessation tends to be more successful among affluent than disadvantaged groups. This means that for stop smoking services there is a trade-off to be had in terms of maximising the number of quitters and reducing socioeconomic inequalities in smoking prevalence. Current performance targets for the national stop smoking services in the UK are set only in terms of numbers of quitters, which does not encourage the adoption of strategies to reduce socioeconomic inequalities in smoking prevalence. DISCUSSION: This paper proposes an assessment framework, which allows the two dimensions of overall reduction in smoking prevalence and reductions of inequalities in smoking prevalence to be assessed together. The framework is used to assess the performance over time of a stop smoking service in Derwentside, a former Primary Care Trust in the North East of England, both in terms of meeting targets for the overall number of quitters and in terms of reducing socioeconomic inequalities in smoking prevalence. The example demonstrates how the proposed assessment framework can be applied in practice given existing records kept by stop smoking services in England and the available information on smoking prevalence at small area level. For Derwentside it is shown that although service expansion was successful in increasing the overall number of quitters, the service continued to exacerbate inequality in smoking prevalence between deprived and affluent wards. SUMMARY: The Secretary of State for Health in the UK has warned about the dangers of health promotion services and messages being taken up more readily by the better-off, thus exacerbating health inequalities. Because smokers from affluent backgrounds are more successful at quitting than those living in deprived circumstances, it is important to build an equity element into the monitoring of individual stop smoking services. Otherwise the danger highlighted by the Secretary of State for Health will go undetected and unaddressed.

Profiling cytochrome P450 expression in ovarian cancer: identification of prognostic markers.

PURPOSE: The cytochromes P450 are a multigene family of enzymes with a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs and biologically active endogenous compounds. The purpose of this study was to define the cytochrome P450 profile of ovarian cancer and identify novel therapeutic targets and establish the prognostic significance of expression of individual cytochrome P450s in this type of cancer. EXPERIMENTAL DESIGN: Immunohistochemistry for a panel of 23 cytochrome P450s and cytochrome P450 reductase was done on an ovarian cancer tissue microarray consisting of 99 primary epithelial ovarian cancers, 22 peritoneal metastasis, and 13 normal ovarian samples. The intensity of immunoreactivity in each sample was established by light microscopy. RESULTS: In primary ovarian cancer, several P450s (CYP1B1, CYP2A/2B, CYP2F1, CYP2R1, CYP2U1, CYP3A5, CYP3A7, CYP3A43, CYP4Z1, CYP26A1, and CYP51) were present at a significantly higher level of intensity compared with normal ovary. P450 expression was also detected in ovarian cancer metastasis and CYP2S1 and P450 reductase both showed significantly increased expression in metastasis compared with primary ovarian cancer. The presence of low/negative CYP2A/2B (log rank = 7.06, P = 0.008) or positive CYP4Z1 (log rank = 6.19, P = 0.01) immunoreactivity in primary ovarian cancer were each associated with poor prognosis. Both CYP2A/2B and CYP4Z1 were also independent markers of prognosis. CONCLUSIONS: The expression profile of individual P450s has been established in ovarian cancer. Several P450s show increased expression in ovarian cancer and this provides the basis for developing P450-based therapeutics in ovarian cancer. Expression of CYP2A/2B or CYP4Z1 in primary ovarian cancer were independent markers of prognosis.

Neoadjuvant chemotherapy in breast cancer: significantly enhanced response with docetaxel.

PURPOSE: To compare the efficacy of neoadjuvant (NA) docetaxel (DOC) with anthracycline-based therapy and determine the efficacy of NA DOC in patients with breast cancer initially failing to respond to anthracycline-based NA chemotherapy (CT). PATIENTS AND METHODS: Patients with large or locally advanced breast cancer received four pulses of cyclophosphamide 1,000 mg/m(2), doxorubicin 50 mg/m(2), vincristine 1.5 mg/m(2), and prednisolone 40 mg (4 x CVAP) for 5 days. Clinical tumor response was assessed. Those who responded (complete response [CR] or partial response [PR]) were randomized to receive further 4 x CVAP or 4 x DOC (100 mg/m(2)). All nonresponders received 4 x DOC. RESULTS: One hundred sixty-two patients were enrolled; 145 patients completed eight cycles of NA CT. One hundred two patients (66%) achieved a clinical response (PR or CR) after 4 x CVAP. After randomization, 50 patients received 4 x CVAP and 47 patients received 4 x DOC. In patients who received eight cycles of CT, the clinical CR (cCR) and clinical PR (cPR) (94% v 66%) and pathologic CR (pCR) (34% v 16%) response rates were higher (P =.001 and P =.04) in those who received further DOC. Intention-to-treat analysis demonstrated cCR and cPR (85% v 64%; P =.03) and pCR (31% v 15%; P =.06). Axillary lymph node examination revealed residual tumor in 33% of patients who received 8 x CVAP and 38% of patients who received further DOC. In patients who failed to respond to the initial CVAP, 4 x DOC resulted in a cCR and cPR rate of 55% and a pCR rate of 2%. Forty-four percent of these patients had residual tumor within axillary lymph nodes. CONCLUSION: NA DOC resulted in substantial improvement in responses to DOC.

Neoadjuvant docetaxel in breast cancer: 3-year survival results from the Aberdeen trial.

Over the past 30 years there has been an increased use of neoadjuvant (or primary) chemotherapy for treating patients with breast cancer. However, while it is clear that chemotherapy given in the adjuvant setting after surgery does prolong patients' overall and disease-free survival, the evidence that chemotherapy in the neoadjuvant setting also increases survival remains unproven. In the Aberdeen study, 162 patients with large and locally advanced breast cancer underwent 4 cycles of CVAP (cyclophosphamide/vincristine/doxorubicin/prednisone) primary chemotherapy. Patients with a complete or partial response were then randomized to either 4 further cycles of CVAP or 4 cycles of docetaxel (100 mg/m2). It was shown that the addition of sequential docetaxel (100 mg/m2) to CVAP neoadjuvant chemotherapy resulted in a significantly enhanced clinical response rate (94% vs. 64%) and a substantially increased complete histopathological response rate (34% vs. 16%) when compared to patients receiving CVAP alone. Furthermore, patients receiving docetaxel had an increased breast conservation rate (67% vs. 48%) and an increased survival at a median follow-up of 3 years. It is important to note that this was a small study, and the survival results should be interpreted with caution. The results are encouraging, however, and further studies are urgently required.

A new histological grading system to assess response of breast cancers to primary chemotherapy: prognostic significance and survival.

The clinical and complete pathological response of a primary breast cancer to chemotherapy has been shown to be an important prognostic for survival. However, the majority of patients do not experience a complete pathological response to primary chemotherapy and the significance of lesser degrees of histological response is uncertain and the prognostic significance is unknown. The purpose of this study was to evaluate a new histological grading system to assess response of breast cancers to primary chemotherapy and to determine if such a system has prognostic value.A consecutive series of 176 patients with large (> or =4cm) and locally advanced breast cancers were treated with multimodality therapy comprising primary chemotherapy, surgery, radiotherapy and tamoxifen. All underwent assessment of the primary breast tumour before and after completion of chemotherapy. Residual tumour was excised after completion of chemotherapy (mastectomy or wide local excision with axillary surgery). The removed tissue was assessed and response to chemotherapy graded using a five-point histological grading system based with the fundamental feature being a reduction in tumour cellularity; comparison being made with a pre-treatment core biopsy. All patients were followed up for 5 years or more. Pathological responses were compared to 5 year overall survival and disease-free survival using log rank tests. The overall 5-year survival for all patients was 71%, and 5 year disease free interval was 60%. There was a significant correlation between pathological response using this new grading system and both overall survival (P=0.02) and disease-free interval (P=0.04). In a multivariate analysis of known prognostic factors, the Miller/Payne grading system was an independent predictor of overall patient survival. This grading system, which assesses the histological response to primary chemotherapy, can predict overall survival and disease-free interval in patients with large and locally advanced breast cancers treated with such therapy. The relationship of degree of histological response to overall and disease-free survival has been shown in univariate and multivariate analyses and could potentially have an important role in the clinical management of patients with locally advanced breast cancer undergoing primary chemotherapy.

Total syntheses of (-) epilupinine and (-)-tashiromine using imino-aldol reactions.

Short routes to enantiomerically pure indolizidine and quinolizidine alkaloids have been developed using imino-aldol reactions of enolates derived from phenyl 5-chlorovalerate. High levels of syn selectivity (dr ∼13-16:1) were obtained using lithium enolates of phenyl esters in combination with tert-butylsulfinyl imines. The imino-aldol adducts were deprotected and cyclized to afford (-)-epilupinine ((-)-2) and (-)-tashiromine ((-)-1) in two further steps.

Market access challenges in the EU for high medical value diagnostic tests.

The clinical utility and medico-economic value of several personalized diagnostic tests has been well described in the literature. Development of such tests, including generation of the necessary supportive clinical validation data, is a complex and expensive endeavor. In general, sponsors of such tests lack sufficient clarity on appropriate reimbursement and regulatory pathways to provide the clear development framework necessary to incentivize the required level of investment. In the USA, an imperfect reimbursement paradigm has evolved to accommodate a small number of 'value-priced' laboratory-developed tests, although major structural barriers remain to broader implementation. In Europe, by contrast, there is virtually no precedent for value-based public sector pricing, and even such procedurally based pricing as currently exists is administered by a complex network of largely decentralized bodies. As a consequence, patient access is limited and health-economic savings are not realized. This article explores some of the European market entry barriers, with a focus on reimbursement challenges, and highlights some collaborative proposals to address such.