About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated ( approximately 45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure-function relationships for lymphocyte perforin.

Since the early days of megavoltage Radiation Therapy (RT), the potential of delivering treatment to a sub group of patients in an upright position has been recognized. Compared to lying horizontally, treating patients in an upright position offers potential benefits in terms of patient comfort especially for patients experiencing dyspnoea and saliva accumulation when lying down. Dosimetric benefits can also be gained from changes in the volume and location of lungs and heart in an upright position, which are potentially advantageous for clinical situations including Hodgkin's disease, lung and breast malignancies. Since the 1950's, upright stabilization mechanisms have ranged from standalone chair based apparatus to couch-top attachments with increasingly customizable solutions. The introduction of Computed-Tomography (CT) based three-dimensional (3D) dosimetry in the 1980's−90's necessitated image acquisition in a horizontal position (supine or prone), significantly reducing options for alternative patient positioning and upright techniques. Despite this, upright techniques have still been utilized where clinically indicated for palliative and novel approaches often involving non-standard treatment scenarios. More recently, a small number of centers have reported on specialized equipment capable of acquiring planning data with the patient in a vertical position. The possibility of acquiring planning quality Cone Beam CT (CBCT) on linear accelerators has recently reinvigorated the potential to deliver highly accurate and targeted treatments to patients in an upright position. This paper reflects on the historical applications of upright RT and explores new possibilities for this technology in modern RT departments.

For almost two decades, breast cancer clinical genetics has operated in an environment where a heritable cause of breast cancer susceptibility is identified in the vast minority of women seeking advice about their personal and/or family history of breast and/or ovarian cancer. A new wave of genetic information is upon us that promises to provide an explanation for the greater proportion of current missing heritability of breast cancer. Whilst researchers refine bioinformatic and analytic methodology necessary to interpret the new genetic data, attention needs to be paid to defining appropriate and coordinated pathways for the translation of this information so that it can be applied in clinical genetic services for the benefit of the majority of women who currently have no explanation for their breast cancer susceptibility. The search for additional breast cancer susceptibility genes remains a very active area of research. Exhausting the power of linkage studies that identified BRCA1 and BRCA2, the research community moved to candidate gene studies that led to the identification of ATM, BRIP1, CHEK2, and PALB2 as so-called "moderate-risk" breast cancer susceptibility genes. Mutations in these genes are rare and although early reports suggested that, on average, they are associated with moderate risks of breast cancer; population-based studies have demonstrated that at least some mutations in these genes are associated with breast cancer risks that are comparable to the average risk associated with BRCA2 mutations. The search for additional breast cancer susceptibility genes has now moved onto research platforms applying massively parallel sequencing capable of sequencing whole human exomes and genomes in single instrument runs. These programs are identifying a large number of additional putative breast cancer susceptibility genes, many of which are currently undergoing validation. It is highly anticipated that the remaining missing heritability of breast cancer will be due to mutations in many different genes, each explaining a small proportion of the currently unexplained heritable breast cancer susceptibility. The characterization of PALB2 as a breast cancer susceptibility gene and subsequent research that has refined our understanding of the prevalence and penetrance of heritable mutations in PALB2 offers a precious opportunity to use the data as a model and develop modes of translation that would be appropriate for the anticipated volume of imminent new information.

There remains debate about whether risk-reducing salpingo-oophorectomy (RRSO), which reduces ovarian cancer risk, also reduces breast cancer risk. We examined the association between RRSO and breast cancer risk using a prospective cohort of 17 917 women unaffected with breast cancer at baseline (7.2% known carriers of BRCA1 or BRCA2 mutations). During a median follow-up of 10.7 years, 1046 women were diagnosed with incident breast cancer. Modeling RRSO as a time-varying exposure, there was no association with breast cancer risk overall (hazard ratio [HR] = 1.04, 95% confidence interval [CI] = 0.87 to 1.24) or by tertiles of predicted absolute risk based on family history (HR = 0.68, 95% CI = 0.32 to 1.47, HR = 0.94, 95% CI = 0.70 to 1.26, and HR = 1.10, 95% CI = 0.88 to 1.39, for lowest, middle, and highest tertile of risk, respectively) or for BRCA1 and BRCA2 mutation carriers when examined separately. There was also no association after accounting for hormone therapy use after RRSO. These findings suggest that RRSO should not be considered efficacious for reducing breast cancer risk.

The transformation of follicular lymphoma to an aggressive lymphoma is a well-recognised complication that occurs at a rate of approximately 3 % a year for the first 10 years of observation. Transformation is accompanied by increased risk of refractoriness and a poor expectation of survival. Genetic and epigenetic triggers for transformation have been described. Prior to routine use of rituximab, transformed lymphoma was managed in a fashion similar to that for de novo diffuse large B-cell lymphoma, with generally poor results. Rituximab appears to have improved outcomes. Some centres, including our own, use high-dose chemotherapy with stem cell transplantation as consolidation for those with responsive disease. Here, we focus on transformed follicular lymphoma, and provide an overview of the current literature and our approach to management.

The integrative control of diverse biological processes such as proliferation, differentiation, apoptosis and metabolism is essential to maintain cellular and tissue homeostasis. Disruption of these underlie the development of many disease states including cancer and diabetes, as well as many of the complications that arise as a consequence of aging. These biological outputs are governed by many cellular signaling networks that function independently, and in concert, to convert changes in hormonal, mechanical and metabolic stimuli into alterations in gene expression. First identified in Drosophila melanogaster as a powerful mediator of cell division and apoptosis, the Hippo signaling pathway is a highly conserved regulator of mammalian organ size and functional capacity in both healthy and diseased tissues. Recent studies have implicated the pathway as an effector of diverse physiological cues demonstrating an essential role for the Hippo pathway as an integrative component of cellular homeostasis. In this review, we will: (a) outline the critical signaling elements that constitute the mammalian Hippo pathway, and how they function to regulate Hippo pathway-dependent gene expression and tissue growth, (b) discuss evidence that shows this pathway functions as an effector of diverse physiological stimuli and

BACKGROUND: It has been hypothesised that increased VEGF-D expression may be an independent prognostic factor for endometrial cancer progression and lymph node metastasis; however, the mechanism by which VEGF-D may promote disease progression in women with endometrial cancer has not been investigated. Our aim was to describe the distribution of lymphatic vessels in mouse uterus and to examine the effect of VEGF-D over-expression on these vessels in a model of endometrial cancer. We hypothesised that VEGF-D over-expression would stimulate growth of new lymphatic vessels into the endometrium, thereby contributing to cancer progression. METHODS: We initially described the distribution of lymphatic vessels (Lyve-1, podoplanin, VEGFR-3) and VEGF-D expression in the mouse uterus during the estrous cycle, early pregnancy and in response to estradiol-17beta and progesterone using immunohistochemistry. We also examined the effects of VEGF-D over-expression on uterine vasculature by inoculating uterine horns in NOD SCID mice with control or VEGF-D-expressing 293EBNA tumor cells. RESULTS: Lymphatic vessels positive for the lymphatic endothelial cell markers Lyve-1, podoplanin and VEGFR-3 profiles were largely restricted to the connective tissue between the myometrial circular and longitudinal muscle layers; very few lymphatic vessel profiles were observed in the endometrium. VEGF-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium), although expression was generally low. VEGF-D immunoexpression was slightly but significantly higher in estrus relative to diestrus; and in estradiol-17beta treated mice relative to vehicle or progesterone treated mice. The presence of VEGF-D over-expressing tumor cells did not induce endometrial lymphangiogenesis, although changes were observed in existing vessel profiles. For myometrial lymphatic and endometrial blood vessels, the percentage of profiles containing proliferating endothelial cells, and the cross sectional area of vessel profiles were significantly increased in response to VEGF-D in comparison to control tumor cells. In contrast, no significant changes were noted in myometrial blood vessels. In addition, examples of invading cells or tumor emboli were observed in mice receiving VEGF-D expressing 293EBNA cells. CONCLUSIONS: These results illustrate that VEGF-D over-expression has differential effects on the uterine vasculature. These effects may facilitate VEGF-D's ability to promote endometrial cancer metastasis and disease progression.

A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in significantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efficacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

Expression of hypoxia-inducible factor (HIF)1α increases the risk of castrate-resistant prostate cancer (CRPC) and metastases in patients on androgen deprivation therapy (ADT) for prostate cancer (PC). We aimed to investigate the effects of nonspecific HIF1α inhibitors (Digoxin, metformin, and angiotensin-2 receptor blockers) on development of CRPC and metastases while on ADT. A retrospective review of prospectively collected medical records was conducted of all men who had continuous ADT as first-line therapy for CRPC at the Austin Hospital from 1983 to 2011. Association between HIF1α inhibitor medications and time to develop CRPC was investigated using actuarial statistics. Ninety-eight patients meeting the criteria were identified. Eighteen patients (21.4%) were treated with the nonspecific HIF1α inhibitors. Both groups had similar characteristics, apart from patients on HIF1α inhibitors being older (70 years vs. 63.9 years). The median CRPC-free survival was longer in men using HIF1α inhibitors compared to those not on inhibitors (6.7 years vs. 2.7 years, P = 0.01) and there was a 71% reduction in the risk of developing CRPC (HR 0.29 [95% CI 0.10-0.78] P = 0.02) after adjustment for Gleason score, age, and prostate-specific antigen (PSA). The median metastasis-free survival in men on HIF1α inhibitors was also significantly longer compared to those on no inhibitors (5.1 years vs. 2.6 years, P = 0.01) with an 81% reduction in the risk of developing metastases (HR 0.19 [CI 0.05-0.76] P = 0.02) after adjustment for Gleason score, age, and PSA. Nonspecific HIF1α inhibitors appear to increase the progression-free survival and reduce the risk of developing CRPC and metastases in patients on continuous ADT.

Objective The management of low-risk febrile neutropenia (FN) patients through ambulatory programs has demonstrated comparative safety and effectiveness to in-patient strategies. However, there is limited evidence of benefits of changing practice, particularly on a national scale. The aim of this study was to estimate costs and benefits of the program over a 10-year time horizon. Methods A comparative cost analysis from a health system perspective was performed, comparing costs and length of stay (LOS) of patients enrolled in an ambulatory program to a historical cohort who did not receive the program. Generalised linear models were used for analysis and bootstrapped to account for uncertainty. National data of identified FN admissions were used to inform future projections, with varying proportions of low-risk patients and eligibility for the ambulatory program. Results The overall LOS for patients in ambulatory cohort was 1.9 days shorter (95% confidence interval (CI) 1.0-2.8 days), a 50% reduction in in-patient bed-days. Although patients in the ambulatory cohort incurred additional costs due to care received outside hospital (mean (± s.d.) A$828.03 ± 124.30), the mean total cost incurred remained substantially lower than that of the historical cohort (A$2979 lower; 95% CI A$772-5391). On a national scale, this could translate into A$62.7 million in costs averted and 41347 bed-days saved over 10 years if the low-risk prediction rate and eligibility for ambulatory programs remained at currently observed rates. Conclusions The wider implementation of a safe and effective ambulatory program to manage low-risk FN patients can result in significant return-on-investment for the healthcare system by eliminating avoidable costs due to unnecessary lengthy hospital admissions. What is known about the topic? There is strong evidence demonstrating out-patient treatment of low-risk FN patients to be an effective and cost-effective strategy compared with continued in-patient hospitalisation. What does this paper add? This study demonstrates the sustainability of the ambulatory program in ensuring cost benefits and in-patient beds through real-life implementation data. It also provides evidence of the substantial cost and bed-days potentially averted when the cost savings and difference in LOS are estimated on a national scale over a 10-year time horizon. What are the implications for practitioners? The management of low-risk FN patients through ambulatory or out-patient programs is a safe and effective approach. There is strong evidence demonstrating the likely cost savings and considerable bed-days saved, which can be reallocated to meet other medical demands.