Single-cell RNA sequencing identifies molecular targets associated with poor in vitro maturation performance of oocytes collected from ovarian stimulation.

STUDY QUESTION: What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them? SUMMARY ANSWER: The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes. WHAT IS KNOWN ALREADY: rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM. STUDY DESIGN, SIZE, DURATION: We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-µM gallic acid on their maturation rate. MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/ß-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%. LARGE SCALE DATA: Raw data from this study can be accessed through GSE158539. LIMITATIONS, REASONS FOR CAUTION: In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.

Effects of the combined administration of vitamins C and E on the oxidative stress status and programmed cell death pathways after experimental spinal cord injury.

STUDY DESIGN: Experimental, controlled, animal study. OBJECTIVES: To assess the effects of vitamins C and E (VCE) treatment on oxidative stress and programmed cell deaths after rat spinal cord injury (SCI), as well as functional recovery. SETTING: Taiwan. METHODS: Fifty-four Sprague-Dawley rats were used for the experimental procedure. In the sham group, laminectomy at T10 was performed, followed by impactor contusion of the spinal cord. In the control group, only a laminectomy was performed without contusion. Oxidative stress status was assessed by measuring the spinal cord tissue content of superoxide dismutase (SOD) and gluthatione peroxidase (GSH-Px) activities. We also evaluated the effects of combined VCE treatment using western blot to analyze expression of cleaved caspase-3 and microtubule-associated protein light chain 3 (LC3), and the Basso, Beattie and Bresnahan (BBB) scale to evaluate functional outcomes. RESULTS: Combined treatment of VCE significantly counteracted the effects of spinal cord contusion on oxidative stress represented by activities of SOD and GSH-Px (P<0.05). The VCE treatment also significantly enhanced LC3-II expression and decreased cleaved caspase-3 compared with the sham (P<0.05). Furthermore, BBB scores significantly improved in the VCE-treated group compared with the sham group (on day 14 and 28 after SCI; P<0.05). CONCLUSIONS: The combined administration of VCE was clearly capable of modulating the antioxidant effects, and of reducing apoptosis and increasing autophagy at the lesion epicenter leading to an improved functional outcome. Use of such clinically ready drugs could help earlier clinical trials in selected cases of human SCIs.

Global Health System Resilience during Encounters with Stressors - Lessons Learnt from Cancer Services during the COVID-19 Pandemic.

AIMS: The protracted COVID-19 pandemic has overwhelmed health systems globally, including many aspects of cancer control. This has underscored the multidimensional nature of cancer control, which requires a more comprehensive approach involving taking a wider perspective of health systems. Here, we investigated aspects of health system resilience in maintaining cancer services globally during the COVID-19 pandemic. This will allow for health systems to be resilient to different types of system stressors/shocks in the future, to allow cancer care to be maintained optimally. MATERIALS AND METHODS: Using the World Health Organization health system framework (capturing aspects of service delivery, health workforce, information, medical products, vaccines and technologies, financing and governance and leadership), we carried out a comparative analysis of the impact of COVID-19 and the synthesis of the findings in responses in cancer care in 10 countries/jurisdictions across four continents comprising a wide diversity of health systems, geographical regions and socioeconomic status (China, Colombia, Egypt, Hong Kong SAR, Indonesia, India, Singapore, Sri Lanka, UK and Zambia). A combination of literature and document reviews and interviews with experts was used. RESULTS: Our study revealed that: (i) underlying weaknesses of health systems before the pandemic were exacerbated by the pandemic (e.g. economic issues in low- and middle-income countries led to greater shortage of medication and resource constraints compounded by inadequacies of public financing and issues of engagement with stakeholders and leadership/governance); (ii) no universal adaptive strategies were applicable to all the systems, highlighting the need for health systems to design emergency plans based on local context; (iii) despite the many differences between health systems, common issues were identified, such as the lack of contingency plan for pandemics, inadequate financial policies for cancer patients and lack of evidence-based approaches for competing priorities of cancer care/pandemic control. CONCLUSION: We identified four key points/recommendations to enhance the resilient capacity of cancer care during the COVID-19 pandemic and other system stressors: (i) effective pandemic control approaches in general are essential to maintain the continuity of cancer care during the emergency health crises; (ii) strong health systems (with sufficient cancer care resources, e.g. health workforce, and universal health coverage) are fundamental to maintain quality care; (iii) the ability to develop response strategies and adapt to evolving evidence/circumstances is critical for health system resilience (including introducing systematic, consistent and evidence-based changes, national support and guidance in policy development and implementation); (iv) preparedness and contingency plans for future public health emergencies, engaging the whole of society, to achieve health system resilience for future crises and to transform healthcare delivery beyond the pandemic.

Noninvasive investigation of asymmetrically conjoined tripus twins with features of rachipagus, parapagus dicephalus, and cephalopagus.

A hypothetical mechanism for conjoined twinning postulated by Spencer ([2003] Developmental Malformations and Clinical Implications, Baltimore: Johns Hopkins University Press, p 1-476) suggests that, after separation, monovular twins fuse in one of eight predictable homologous sites. The tripus fetal specimen under study embodies characteristics of three types therefore preventing it from classification into a simple variant of any one of the eight twin types described by Spencer. The aim of this study was to reveal internal structural anomalies of the fetal specimen by using magnetic resonance imaging and computerized tomography. Dorsally appended to the primary twin is a secondary head mass (brain tissue and ocular globe) and two spinal columns converging at T4/T5, suggesting rachipagus twinning. The ventral orientation of the secondary twin's (right lateral) lower limb suggests parapagus twinning. The caudal divergence of the spinal columns and the presence of a secondary hemipelvis, separate from the primary pelvis, suggest cephalopagus twinning. Measurements of the long bones indicate a gestational age of ∼20-23 weeks. Secondary malformations of the primary fetal body include anencephaly, cleft palate, renal agenesis, decreased left ventricular outflow, and a prematurely terminating descending aorta. This study demonstrates the possibility of using current imaging techniques to study very old, formalin-preserved human material for documentation and scientific discussion without destroying the specimen, thus keeping it intact for posterity.

Parovarian tumors of borderline malignancy.

The incidence of a parovarian tumor is 10-20% of all uterine adnexal masses, however, it is benign in most cases, and a borderline or malignant tumor is extremely rare. The classification of disease stage and treatment is still controversial owing to its scarcity. We have managed one mucinous and two serous cystadenomas of borderline malignancy originating from paraovarian cysts in our institute over ten year. We report and discuss the cases herein.

Combination atezolizumab, bevacizumab, pemetrexed and carboplatin for metastatic EGFR mutated NSCLC after TKI failure.

INTRODUCTION: Acquired resistance to TKI is an important unmet need in the management of EGFR mutated lung cancer. Recent clinical trial IMPower150 suggested that combination approach with VEGF inhibitor, check point inhibitor immunotherapy and platinum-based chemotherapy was effective in oncogene driven lung cancer. The current trial examined the efficacy of a modified regimen in an EGFR mutated cohort. METHODS: An open-labelled, single arm, phase II study was conducted in patients with EGFR mutated NSCLC who had progressed on at least one EGFR TKI. For those with T790M mutation, radiological progression on osimertinib was required for enrolment. Patients were treated with combination atezolizumab (1200 mg), bevacizumab (7.5 mg/kg), pemetrexed (500 mg/m2) and carboplatin (AUC 5) given once every 3 weeks until progression. RESULTS: Forty patients were enrolled. Median age was 62 (range 45-76) years. More than one half (23/40, 57.5%) had progressed on osimertinib. PD-L1 expression was < 1% in 52.5%. Median follow-up time was 17.8 months. ORR was 62.5%. Median PFS was 9.4 months (95% CI: 7.6 - 12.1). One year OS was 72.5% (95% CI: 0.56-0.83). Treatment related grade 3 or above adverse events (AE) occurred in 37.5% (15/40). Immune-related AE occurred in 32.5% (13/40) patients. Quality of life measures of function and symptoms did not change significantly throughout the course of treatments. Post-trial rechallenge with EGFR TKI containing regimen resulted in PFS of 5.8 months (95% CI 3.9-10.0 months). CONCLUSION: Combination approach of atezolizumab, bevacizumab, pemetrexed and carboplatin achieved promising efficacy in metastatic EGFR mutated NSCLC after TKI failure. The results were comparable with taxane based regimen of IMPower150 while toxicity profile was improved.

Molecular markers and targeted therapy of skin rejection in composite tissue allotransplantation.

Skin rejection remains a major hurdle in reconstructive transplantation. We investigated molecular markers of skin rejection with particular attention to lymphocyte trafficking. Skin biopsies (n = 174) from five human hand transplant recipients were analyzed for rejection, characteristics of the infiltrate and lymphocytic adhesion markers. The cellular infiltrate predominantly comprised CD3+ T cells. CD68, Foxp3 and indoleamine 2, 3-dioxygenase expression and the CD4/CD8 increased with severity of rejection. Lymphocyte adhesion markers were upregulated upon rejection, intercellular adhesion molecule-1 and E-selectin correlated best with severity of rejection. Guided by the findings, a specific E- and P-selectin inhibitor was investigated for its effect on skin rejection in a rat hind limb allotransplant model. While efomycine M (weekly s.c. injection into the graft) alone had no effect, long-term allograft survival was achieved when combined with antithymocyte globulin and tacrolimus (control group without efomycine M rejected at postoperative day [POD] 61 +/- 1). Upregulation of lymphocyte trafficking markers correlates with severity of skin rejection and time after transplantation in human hand transplantation. Blocking E- and P-selectin in the skin holds potential to significantly prolong limb allograft survival.

Functional stoichiometry of Shaker potassium channel inactivation.

Shaker potassium channels from Drosophila are composed of four identical subunits. The contribution of a single subunit to the inactivation gating transition was investigated. Channels carrying a specific mutation in a single subunit can be labeled in a heterogeneous population and studied quantitatively with scorpion toxin sensitivity as a selection tag. Linkage within a single subunit of a mutation that removes the inactivation gate to a second mutation that affects scorpion toxin sensitivity demonstrates that only a single gate is necessary to produce inactivation. The inactivation rate constant for channels with a single gate was one-fourth that of channels with four gates. In contrast, the rate of recovery from inactivation was independent of the number of gates. It appears that each of the four open inactivation gates in a Shaker potassium channel is independent, but only one of the four gates closes in a mutually exclusive manner.

Total synthesis of the L-hexoses.

Enantiomerically pure polyhydroxylated natural products are synthesized by using a reiterative two-carbon extension cycle consisting of four steps. The generality and efficiency of this methodology are demonstrated in the total synthesis of all eight L-hexoses.

Central retinal artery occlusion: timing and mode of presentation.

BACKGROUND AND PURPOSE: Central retinal artery occlusion (CRAO) is a sudden, frequently irreversible, monocular vision loss, analogous to acute cerebral ischaemia. Thrombolysis may improve visual outcomes, but it is unclear what the acceptable timing of administration should be. We aim to ascertain, through audit, the timing and mode of presentation of individuals with potentially thrombolysable CRAOs. METHODS: A retrospective audit of patients with acute thromboembolic CRAO. RESULTS: Thirty-one patients were identified. Mean time from onset of vision loss to presentation was 31 +/- 65 h. Eighteen patients (58%) presented within 20 h of vision loss, and the majority presented first to a general practitioner. Fifteen patients (48%) were reviewed by an in-hospital ophthalmologist within 20 h of vision loss. The mean delay from the referring source to assessment by an in-hospital ophthalmologist was 5.2 h (median 3.3 h, range 50 min to 24 h). This delay was, on average, shorter for patients referred directly to an ophthalmology clinic. CONCLUSIONS: Just under half (48%) of our cohort of CRAO patients were reviewed by an in-hospital ophthalmologist within the 20-h therapeutic time window for thrombolytic therapy and thus could qualify for inclusion in a randomized controlled trial according to EAGLE inclusion criteria. If thrombolysis is proven to be a feasible and safe treatment in CRAO then public awareness should be raised of the symptoms and an efficient direct referral pathway to an in-hospital ophthalmologist established to aid treatment delivery.

Indoleamine 2,3-dioxygenase and foxp3 expression in skin rejection of human hand allografts.

Human hand transplantation is complicated by skin rejection. To better define the characteristics of infiltrating cells, biopsies from human hand transplants have been investigated for expression of Foxp3 and indoleamine 2,3-dioxygenase (IDO), a key regulatory enzyme to induce T-lymphocyte unresponsiveness. A total of 104 skin biopsies taken from three bilateral hand transplant recipients over 6 years posttransplant were assessed by hematoxylin-eosin histology (graded 1-4b) and immunohistochemistry for IDO and Foxp3 according to a three-grade classification and correlated with the grade of rejection as well as time after transplantation. Overall, rejection ranged between grades 0 and 4a with an average score of 0.94. IDO was expressed in the endothelium independent of rejection. Upon rejection, IDO staining within the cellular infiltrate was significantly increased. Foxp3 in regulatory T cells was mainly found in samples undergoing severe rejection. Expression of IDO and Foxp3 compared well to each other, although the overall expression of Foxp3 was lower when compared to IDO. An increased expression of IDO as well as Foxp3 during rejection late after transplantation was observed. Characteristics of the cellular infiltrate indicate tolerogenic properties of a proportion of the cells and therefore a tendency toward self-limitation of the alloimmune response during skin rejection after hand transplantation.

Integrative Palliative Cancer Care in Hong Kong: An Overview and an Example from the East.

Cancer is the most common cause of mortality worldwide. Although recent advances of multiple modality cancer management have significantly improved the cure and control rates, a significant proportion of patients are still refractory to the standard and available treatments. Early initiation of palliative care can reduce cancer suffering, improve health-related quality of life and possibly prolong survival. It also allows patients and their caretakers to perceive the trajectory of their cancer, so that better and advanced care planning can be contemplated and implemented. The traditional beliefs and perceptions of cancer also differ significantly between the East and the West, which may also affect the preferential approach to palliative care. This review provides an overview of palliative care services in Hong Kong, as compared with other parts of the world. In addition, we shall also explore how cancer perceptions affect the decision-making on palliative care.

A rule-based method for predicting the electrical activation of the heart with cardiac resynchronization therapy from non-invasive clinical data.

BACKGROUND: Cardiac Resynchronization Therapy (CRT) is one of the few effective treatments for heart failure patients with ventricular dyssynchrony. The pacing location of the left ventricle is indicated as a determinant of CRT outcome. OBJECTIVE: Patient specific computational models allow the activation pattern following CRT implant to be predicted and this may be used to optimize CRT lead placement. METHODS: In this study, the effects of heterogeneous cardiac substrate (scar, fast endocardial conduction, slow septal conduction, functional block) on accurately predicting the electrical activation of the LV epicardium were tested to determine the minimal detail required to create a rule based model of cardiac electrophysiology. Non-invasive clinical data (CT or CMR images and 12 lead ECG) from eighteen patients from two centers were used to investigate the models. RESULTS: Validation with invasive electro-anatomical mapping data identified that computer models with fast endocardial conduction were able to predict the electrical activation with a mean distance errors of 9.2 ±â€¯0.5 mm (CMR data) or (CT data) 7.5 ±â€¯0.7 mm. CONCLUSION: This study identified a simple rule-based fast endocardial conduction model, built using non-invasive clinical data that can be used to rapidly and robustly predict the electrical activation of the heart. Pre-procedural prediction of the latest electrically activating region to identify the optimal LV pacing site could potentially be a useful clinical planning tool for CRT procedures.

Advancing Care for Head and Neck Cancers in a Multidisciplinary Tumour Board in the East.

Managing head and neck cancers is an excellent example of the importance of teamwork, with head and neck surgeons, clinical oncologists, radiologists, pathologists and other allied health professionals specialised in this disease site working together. The reliable imaging and dedicated pretreatment work-up entailing the comprehensive anatomical description of tumour involvement by the radiologists, the expertise of surgeons in performing en-bloc gross tumour resection, the uneventful speedy postoperative rehabilitation and recovery by the speech therapists and nutritionists, as well as the dedicated treatment planning of clinical oncologists in delivering precise preoperative or postoperative (chemo)radiotherapy to maximise the therapeutic potentials are the pillars of treatment success. A multidisciplinary tumour board involving all of these key players is essential to provide the highest level of recommendation based on evidence-based medicine and to bring patients new hopes and the best chance of cure. This review illustrates the seamless collaborative teamwork within a well-established multidisciplinary tumour board in managing one of the most intractable cancers in the East, taking enlightenment and inspiration from the West.

Presynaptic actions of opioid receptor agonists in ventromedial hypothalamic neurons in estrogen- and oil-treated female mice.

Estrogens act upon ventromedial hypothalamic (VMH) neurons, and their effects on female arousal and sexual behaviors mediated by VMH neurons involve several neurotransmitters and neuromodulators. Among these are opioid peptides which might be predicted to oppose estrogenic action on VMH because they tend to decrease CNS arousal. Spontaneous excitatory postsynaptic currents were recorded from VMH neurons from 17beta-estradiol- (E, 10 mug/0.1 ml) or oil-treated control ovariectomized (OVX) mice using whole-cell patch-clamp techniques. To examine the impact of opioidergic inputs, recordings of neurons from both treatment groups were obtained in the presence of the general opioid receptor agonist methionine enkephalin-Arg-Phe (MERF, 3 muM), or mu-receptor specific agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO, 1 muM). Compared with oil, E treatment for 48 h significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) without affecting their amplitude. MERF and DAMGO each abolished this E effect, causing significant reductions in sEPSCs. The effect of MERF was abolished by naltrexone (general opioid receptor antagonist, 3 muM) and the effect of DAMGO by d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (mu-opioid receptor selective antagonist, 1 muM); in contrast, kappa- and delta-opioid receptor agonists, U69593 (300 nM) and [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE, 1 muM) respectively, had little effect on the sEPSCs compared with DAMGO. To consider presynaptic vs. postsynaptic effects of opioids, miniature excitatory postsynaptic currents (mEPSCs) were investigated in E- and oil-treated VMH neurons and opioid receptor antagonist effects on mEPSCs were observed. Both MERF and DAMGO reduced the frequency of mEPSCs, but had no effect on their amplitude. Our findings indicate that opioids suppress excitatory synaptic transmissions in VMH neurons primarily through mu-receptors and could thereby decrease sexual arousal in mice.

Colony-stimulating factor-1 requires PI3-kinase-mediated metabolism for proliferation and survival in myeloid cells.

Colony-stimulating factor-1 (CSF-1) is essential for macrophage growth, differentiation and survival. Myeloid cells expressing a CSF-1 receptor mutant (DeltaKI) show markedly impaired CSF-1-mediated proliferation and survival, accompanied by absent signal transducers and activators of transcription 3 (Stat3) phosphorylation and reduced PI3-kinase/Akt activity. Restoring phosphatidylinositol 3-kinase (PI3-kinase) but not Stat3 signals reverses the mitogenic defect. CSF-1-induced proliferation and survival are sensitive to glycolytic inhibitors, 2-deoxyglucose and 3-bromopyruvate. Consistent with a critical role for PI3-kinase-regulated glycolysis, DeltaKI cells reconstituted with active PI3-kinase or Akt are hypersensitive to these inhibitors. CSF-1 upregulates hexokinase II (HKII) expression through PI3-kinase, and PI3-kinase transcriptionally activates the HKII promoter. Moreover, HKII overexpression partially restores mitogenicity. In contrast, Bcl-x(L) expression does not enhance long-term proliferation, although short-term cell death is suppressed in a glycolysis-independent manner. This study identifies robust PI3-kinase activation as essential for optimal CSF-1-mediated mitogenesis in myeloid cells, in part through regulation of HKII and support of glycolysis.

Wyburn-Mason syndrome associated with thyroid arteriovenous malformation: a first case report.

A 31-year-old woman with a prior history of Wyburn-Mason syndrome, complicated previously by a left thalamic intracerebral hemorrhage at age 21, complained of sudden left vision loss due to a central retinal vein occlusion. Angiography revealed a left thyroid arterio-venous malformation (AVM) in addition to ones found intracerebrally. The pathogenesis and embryogenesis of this finding including the management of AVMs in Wyburn-Mason syndrome are discussed.

Anterior ischemic optic neuropathy in moyamoya disease: a first case report.

Neuro-ophthalmological manifestations in moyamoya disease are usually the result of cerebrovascular involvement of the visual pathways. We report a case of ischemic optic neuropathy due to ocular hypoperfusion as a result of moyamoya disease, despite a prior internal to external carotid artery bypass with normal hemisphere perfusion. The blood supply of the optic nerve, a proposed pathogenesis of an anterior ischemic optic neuropathy and complications of the ocular ischemic syndrome are discussed.

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors.

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.

Wide local excision and radiotherapy for the treatment of ductal carcinoma in situ of the breast: the Hong Kong experience.

AIMS: Breast conservation treatment for ductal carcinoma in situ (DCIS) was unpopular in the Chinese population and the outcome was seldom reported. We conducted a single-centre retrospective study to examine the clinical outcome of women in Hong Kong. MATERIALS AND METHODS: Seventy-five Chinese women were treated with wide local excision and radiotherapy for DCIS of the breast between 1994 and 2003. Only 26 (34.7%) women had non-palpable DCIS detected by screening mammograms. All women were treated with whole breast irradiation of 50 Gy in 2 Gy daily fractions, with 50 (66.7%) women receiving an additional electron boost of 10-16 Gy. RESULTS: The median follow-up was 5.1 years (range 2.0-10.7). At the last assessment, four women developed local recurrences, but all remained disease-free after salvage mastectomy. The 5-year actuarial local failure-free rate and cause-specific survival rate were 92.9% (95% confidence interval 89.4-96.4) and 100.0%, respectively. Cosmetic results were rated as good to excellent in all women. On univariate analysis of prognostic factors for local failure, only a close (< or = 2 mm) final resection margin approached statistical significance (hazard ratio 9.108; 95% confidence interval 0.946-87.655; P = 0.056). The 5-year actuarial local failure-free rates for women with a close (< or = 2 mm) final resection margin and women with wider margins were 77.0 and 98.2%, respectively. CONCLUSIONS: Despite geographical and demographic differences, the clinical outcome after wide local excision and radiotherapy for DCIS of the breast in Chinese women is comparable with that in Western series. Efforts are needed to achieve cosmetically acceptable tumour-free margins greater than 2 mm.