Genomic characterization of HER2-positive breast cancer and response to neoadjuvant trastuzumab and chemotherapy-results from the ACOSOG Z1041 (Alliance) trial.

Background: HER2 (ERBB2) gene amplification and its corresponding overexpression are present in 15-30% of invasive breast cancers. While HER2-targeted agents are effective treatments, resistance remains a major cause of death. The American College of Surgeons Oncology Group Z1041 trial (NCT00513292) was designed to compare the pathologic complete response (pCR) rate of distinct regimens of neoadjuvant chemotherapy and trastuzumab, but ultimately identified no difference. Patients and methods: In supplement to tissues from 37 Z1041 cases, 11 similarly treated cases were obtained from a single institution study (NCT00353483). We have extracted genomic DNA from both pre-treatment tumor biopsies and blood of these 48 cases, and performed whole genome (WGS) and exome sequencing. Coincident with these efforts, we have generated RNA-seq profiles from 42 of the tumor biopsies. Among patients in this cohort, 24 (50%) achieved a pCR. Results: We have characterized the genomic landscape of HER2-positive breast cancer and investigated associations between genomic features and pCR. Cases assigned to the HER2-enriched subtype by RNA-seq analysis were more likely to achieve a pCR compared to the luminal, basal-like, or normal-like subtypes (19/27 versus 3/15; P = 0.0032). Mutational events led to the generation of putatively active neoantigens, but were overall not associated with pCR. ERBB2 and GRB7 were the genes most commonly observed in fusion events, and genomic copy number analysis of the ERBB2 locus indicated that cases with either no observable or low-level ERBB2 amplification were less likely to achieve a pCR (7/8 versus 17/40; P = 0.048). Moreover, among cases that achieved a pCR, tumors consistently expressed immune signatures that may contribute to therapeutic response. Conclusion: The identification of these features suggests that it may be possible to predict, at the time of diagnosis, those HER2-positive breast cancer patients who will not respond to treatment with chemotherapy and trastuzumab. ClinicalTrials.gov identifiers: NCT00513292, NCT00353483.

The yield of colorectal cancer among fast track patients with normocytic and microcytic anaemia.

INTRODUCTION: We receive fast track referrals on the basis of iron deficiency anaemia (IDA) for patients with normocytic anaemia or for patients with no iron studies. This study examined the yield of colorectal cancer (CRC) among fast track patients to ascertain whether awaiting confirmation of IDA is necessary prior to performing bowel investigations. METHODS: A review was undertaken of 321 and 930 consecutive fast track referrals from Centre A and Centre B respectively. Contingency tables were analysed using Fisher's exact test. Logistic regression analyses were performed to investigate significant predictors of CRC. RESULTS: Overall, 229 patients were included from Centre A and 689 from Centre B. The odds ratio for microcytic anaemia versus normocytic anaemia in the outcome of CRC was 1.3 (95% confidence interval [CI]: 0.5-3.9) for Centre A and 1.6 (95% CI: 0.8-3.3) for Centre B. In a logistic regression analysis (Centre B only), no significant difference in CRC rates was seen between microcytic and normocytic anaemia (adjusted odds ratio: 1.9, 95% CI: 0.9-3.9). There was no statistically significant difference in the yield of CRC between microcytic and normocytic anaemia (p=0.515, Fisher's exact test) in patients with anaemia only and no colorectal symptoms. Finally, CRC cases were seen in both microcytic and normocytic groups with or without low ferritin. CONCLUSIONS: There is no significant difference in the yield of CRC between fast track patients with microcytic and normocytic anaemia. This study provides insufficient evidence to support awaiting confirmation of IDA in fast track patients with normocytic anaemia prior to requesting bowel investigations.

Alterations of BRAF and HIPK2 loci predominate in sporadic pilocytic astrocytoma.

OBJECTIVE: Independent studies have previously demonstrated that both the HIPK2 and BRAF genes are amplified and rearranged, respectively, in pilocytic astrocytomas (PAs). The purpose of this study was to further investigate the frequency of BRAF and HIPK2 alterations in PAs, the concordance of these events, and their relationship to clinical phenotype. METHODS: We performed extensive characterization by array-based copy number assessment (aCGH), HIPK2 copy number analysis, and BRAF rearrangement and mutation analysis in a set of 79 PAs, including 9 tumors from patients with neurofibromatosis type 1 (NF1). RESULTS: We identified 1 of 3 previously identified BRAF rearrangements in 42/70 sporadic PAs. An additional 2 tumors with no rearrangement also exhibited BRAF mutation, including a novel 3-base insertion. As predicted from the genomic organization at this locus, 22/36 tumors with BRAF rearrangement also exhibited corresponding HIPK2 amplification. However, 14/36 tumors with BRAF rearrangement had no detectable HIPK2 gene amplification and 6/20 tumors demonstrated HIPK2 amplification without apparent BRAF rearrangement or mutation. Only 12/70 PAs lacked detectable BRAF or HIPK2 alterations. Importantly, none of the 9 PA tumors from NF1 patients exhibited BRAF rearrangement or mutation. CONCLUSIONS: BRAF rearrangement represents the most common genetic alteration in sporadic, but not neurofibromatosis type 1-associated, pilocytic astrocytomas (PAs). These findings implicate BRAF in the pathogenesis of these common low-grade astrocytomas in children, and suggest that PAs arise either from NF1 inactivation or BRAF gain of function.

Loss of Nkx3.1 leads to the activation of discrete downstream target genes during prostate tumorigenesis.

The expression of NKX3.1, a transcriptional regulator and tumor suppressor gene in prostate cancer, is downregulated during early stages of prostate tumorigenesis. However, little is known of the alterations in gene expression that occur as a result of this event. We combined laser capture microdissection and gene expression profiling to analyse the molecular consequences of Nkx3.1 loss during prostate cancer initiation using Nkx3.1-deficient mice. This analysis identified a cohort of genes (loss-of-Nkx3.1 signature) that are aberrantly overexpressed during loss-of-Nkx3.1-driven tumor initiation. We studied the expression of these genes in independent loss-of-Pten and c-myc overexpression prostate adenocarcinoma mouse models. Nkx3.1 expression is lost in prostate epithelial proliferation in both of these mouse models. However, Nkx3.1 loss is an early event of tumor development in the loss-of-Pten model, whereas it occurs at later stages in c-myc transgenic mice. A number of genes of the loss-of-Nkx3.1 signature, such as clusterin and quiescin Q6, are highly expressed in prostatic hyperplasia and intraepithelial neoplasia (PIN) lesions that also lack Nkx3.1 in the Pten-deficient prostate, but not in similar lesions in the c-myc transgenic model. Meta-analysis of multiple prostate cancer gene expression data sets, including those from loss-of-Nkx3.1, loss-of-Pten, c-myc overexpression and constitutively active Akt prostate cancer models, further confirmed that genes associated with the loss-of-Nkx3.1 signature integrate with PTEN-AKT signaling pathways, but do not overlap with molecular changes associated with the c-myc signaling pathway. In human prostate tissue samples, loss of NKX3.1 expression and corresponding clusterin overexpression are co-localized at sites of prostatic inflammatory atrophy, a possible very early stage of human prostate tumorigenesis. Collectively, these results suggest that the molecular consequences of NKX3.1 loss depend on the epithelial proliferative stage at which its expression is lost, and that alterations in the PTEN-AKT-NKX3.1 axis are important for prostate cancer initiation.

High-resolution, dual-platform aCGH analysis reveals frequent HIPK2 amplification and increased expression in pilocytic astrocytomas.

Pilocytic astrocytomas (PAs, WHO grade I) are the most common brain tumors in the pediatric and adolescent population, accounting for approximately one-fifth of central nervous system tumors. Because few consistent molecular alterations have been identified in PAs compared to higher grade gliomas, we performed array comparative genomic hybridization using two independent commercial array platforms. Although whole chromosomal gains and losses were not observed, a 1-Mb amplified region of 7q34 was detected in multiple patient samples using both array platforms. Copy-number gain was confirmed in an independent tumor sample set by quantitative PCR, and this amplification was correlated to both increased mRNA and protein expression of HIPK2, a homeobox-interacting protein kinase associated with malignancy, contained within this locus. Furthermore, overexpression of wild-type HIPK2, but not a kinase-inactive mutant, in a glioma cell line conferred a growth advantage in vitro. Collectively, these results illustrate the power and necessity of implementing high-resolution, multiple-platform genomic analyses to discover small and subtle, but functionally significant, genomic alterations associated with low-grade tumor formation and growth.

Matrilin-2 expression distinguishes clinically relevant subsets of pilocytic astrocytoma.

Using whole genome expression microarray technology to discover clinically relevant biomarkers for pilocytic astrocytoma (PA), the authors identified matrilin-2 as a unique mRNA overexpressed in PA. Matrilin-2 protein expression was similarly elevated in the majority of sporadic PA, but in only one neurofibromatosis 1-associated PA with an unusually aggressive clinical phenotype. These results suggest that matrilin-2 may be a specific and clinically useful biomarker for discriminating between indolent and clinically aggressive PA.

Determination of the pressure required to cause mitral valve failure.

A method has been developed for applying water pressure to a closed mitral valve on the side corresponding to the heart's left ventricle. The pressure is increased until fluid flows through the valve, i.e. until it fails. A specific dissection technique has been developed to produce a specimen with two annular rings, mitral annulus and papillary muscle annulus. Since the valve is maintained intact, with its leaflets attached to papillary muscles by the chordae tendineae, this method allows the effects of ruptured chordae and their surgical repair or replacement to be assessed in vitro. The chamber that holds the valve supports both the mitral annulus and papillary muscle annulus of the specimen. The mitral annulus is sutured onto rubber sheeting held in the chamber. The papillary muscle annulus is held in place by a Perspex support. The main part of the apparatus consists of a water pump connected through flexible tubing to the chamber that holds the valve in place. The pressure at failure is measured using a pressure transducer. Preliminary experiments demonstrate that anterior leaflet marginal chordae, but not strut chordae, are vital to valve function. Posterior leaflet chordae have been found to be important for valve competence.

Stress avoidance in a common annual: reproductive timing is important for local adaptation and geographic distribution.

Adaptation to local environments may be an important determinant of species' geographic range. However, little is known about which traits contribute to adaptation or whether their further evolution would facilitate range expansion. In this study, we assessed the adaptive value of stress avoidance traits in the common annual Cocklebur (Xanthium strumarium) by performing a reciprocal transplant across a broad latitudinal gradient extending to the species' northern border. Populations were locally adapted and stress avoidance traits accounted for most fitness differences between populations. At the northern border where growing seasons are cooler and shorter, native populations had evolved to reproduce earlier than native populations in the lower latitude gardens. This clinal pattern in reproductive timing corresponded to a shift in selection from favouring later to earlier reproduction. Thus, earlier reproduction is an important adaptation to northern latitudes and constraint on the further evolution of this trait in marginal populations could potentially limit distribution.

Wire tension in the Ilizarov system: accuracy of the wire-tensioning device.

The Ilizarov fixator consists of tensioned wires that attach bone segments to a modular frame. The aim of this study was to establish the accuracy and precision of the wire-tensioning device supplied with the Ilizarov external fixation system. The device was used to tension a wire in direct opposition to a calibrated load cell. Five subjects tested three devices, at each of their four tension settings, in two separate sessions. Subjects could not see the true tension during the test. There were significant differences between the results for different subjects (p < 0.01) and instruments (p < 0.01) but not for different tension settings or between the two sessions. Overall mean measured tensions were 4.9 per cent (standard deviation, 4.4 per cent) below intended values. Tensions obtained at the maximum edge (completely occluded) on the scale markings were significantly (p < 0.001) closer to the nominal values (mean discrepancy, 3.6 per cent) than those at the minimum edge (mean discrepancy, 17.6 per cent). Several factors influence wire tension. Tensioning devices are not identical and the results obtained with them depend on the user. If the scale markings are completely occluded, the discrepancy between intended and actual tensions of around 5 per cent is likely to be adequate for clinical practice since surgeons do not select the most suitable tension following quantitative data assessment, but rather it is a judgement based on surgical experience and consideration for the patient weight and expected level of activity.

A comparison of the torsional performance of stainless steel and titanium alloy tibial intramedullary nails: a clinically relevant approach.

In recent years there has been a tendency to design and manufacture intramedullary nails from titanium alloy rather than from stainless steel. The aim of this project was to compare the torsional performance of one manufacturers standard stainless steel and titanium alloy tibial intramedullary nails, using their distal locking screw holes and dedicated cross screws to secure each nail distally. A custom built test rig and materials testing machine were used to determine the torsional rigidity of the nails. Theory was used to calculate the torsional rigidity of the central parts of each nail. From the mechanical testing, the mean torsional rigidity of the titanium alloy nail system was 40.9 N m2 while that of the stainless steel nail system was 34.6 N m2, for all distal interlocking screw positions tested. Based on theoretical calculations the torsional rigidity of the central part of the nail was 83 N m2 for the stainless steel nail and 66 N m2 for the titanium alloy nail. This study shows the importance of using the distal locking screw holes and dedicated cross screws to secure intramedullary nails during mechanical testing so that clinically relevant results are obtained about the whole nail system and not just the nail.

Synchronously pumped CdSe optical parametric oscillator in the 9-10 microm region.

Continuous mode-locked operation of a singly resonant, synchronously pumped optical parametric oscillator (SPOPO) based on CdSe has produced idler output tuned over the range of 9.1-9.7 microm, the longest wavelength generated so far to our knowledge from a SPOPO. Average idler powers as high as approximately 70 mW are generated in the crystal. Tandem pumping with a diffraction-grating-tuned parametric oscillator in periodically poled lithium niobate provides a convenient and agile means of tuning the noncritically phase-matched CdSe device. The absence of any detrimental thermal effects in the CdSe crystal suggests that significant further power scaling should be possible, with idler tuning ranges extendable to cover 8-12 microm.

Yielding of the clamped-wire system in the Ilizarov external fixator.

This study demonstrates that the clamped-wire system used to suspend bones within an Ilizarov external fixator yields when the perpendicular load exceeds 50 N per wire. Cyclic loading was applied to tensioned wires clamped within an Ilizarov ring component, with steadily increasing load amplitude. Wires were tested at four initial tension settings. The amount of energy lost within the clamped-wire system per load cycle was calculated for every test. The results showed that there was a consistent trend to increasing non-recoverable energy loss per load cycle when peak loads exceed 50 N for all initial tension settings. A finite element (FE) model replicating the experimental conditions was performed to investigate the levels of stress within the loaded wires. The FE model analyses showed that high stresses were generated in the wires close to the clamping sites, and that the stress levels could reasonably be expected to exceed the material yield stress when loaded to about 55 N, for all initial tension settings. The results show that material yield, accompanied by some wire slippage through the clamps, is responsible for system yield, in agreement with previous studies. Although the initial wire tension has an appreciable effect on the wire stiffness, it did not affect the elastic load range of the clamped-wire system. To prevent yield of the clamped-wire system in practice, the fixator should be assembled with sufficient wires to ensure that the load transmitted to each wire by the patient does not exceed 50 N.

The effect of clamping a tensioned wire: implications for the Ilizarov external fixation system.

This study demonstrates that clamping a tensioned wire can cause a reduction in wire tension. Tension (about 1275 N) was applied to a wire that was subsequently clamped, using cannulated bolts, to the steel half-ring of an Ilizarov external fixator. The tension in the wire was monitored before, during and after clamping. The apparatus was disassembled and the deformations in the wire caused by the clamps were measured. This experiment was repeated 15 times. When the wire was clamped to the frame, the wire tension was reduced by 22 +/- 7 per cent (mean +/- standard deviation, SD). The drop in wire tension was linearly correlated (r = 0.96; p < 0.001) with the deformation caused by the bolts. A finite element (FE) model of the wire was also constructed. The model was pre-stressed (tensioned), and the clamping effect replicated. This analysis showed that clamping the wire could be considered to squeeze the wire outwards (like toothpaste from a tube) and so reduce its tension during fixator assembly. To assess the magnitude of this effect in the clinical situation, the FE model analysis was repeated to replicate clamping a 1.8-mm-diameter wire to a 180-mm-diameter steel Ilizarov ring component. The analysis showed that for these conditions the tension reduced by 8-29 per cent. The results of this study highlight a general engineering problem: how can a tensioned wire be secured to a structure without an appreciable loss of tension? If the performance of the structure depends on the wire tension, this performance will change when the wire is secured.

Finite element analysis of stress around a sternum screw used to prevent sternal dehiscence after heart surgery.

The sternum screw has been proposed as a means of preventing sternal dehiscence, following heart surgery, by increasing the contact area between the wire used to close the median sternotomy and the surrounding bone; as a result, the contact stress is reduced. A finite element model was constructed of a cylindrical wire or screw passing through a block of sternum which consisted of cancellous bone sandwiched within a cortical shell. The thickness of the cortical shell and the material properties of bone were varied between reasonable values. The stress distribution in the sternum was calculated for each model when the wire was subjected to a tension (250 N) which would be required for six wires to withstand a strong cough (40 kPa). Results were validated by comparison with a simple analytical model in which the bone and wire were considered incompressible. They show that the screw reduces the contact stress to almost one-seventh of its value when wire is used alone. Contact stresses are especially high if the cortical shell is thin. The high stress in the bone around a screw falls off within a few millimetres. As a result, no problems are anticipated in placing six screws in each half-sternum so that the sternotomy may be closed with the usual six wires.

Peanut hull pellets as a single use sorbent for the capture of Cu(II) from wastewater.

This paper investigates the ability of peanut hulls and peanut hull pellets to adsorb copper from dilute aqueous metal ion solutions in batch and fixed bed systems. The kinetics of copper uptake onto the media have been investigated in batch systems and the influence of pH and particle size on the rate and extent of copper capture determined. The Langmuir and Freundlich isotherm theories were determined; the Langmuir model was found to best represent the equilibrium isotherm data. In normalized kinetic tests at least 75% of copper removal occurred within the first 20 min; 92% removal was effected within the first 50 min. The rate of uptake was optimum within the pH range 5-7.5, and media capacities remained relatively constant at a pH above 4.0. Bench-scale column studies were performed using peanut hull pellets. The overall capacity of pelletized peanut hulls was higher than for unmodified peanut hulls. Due to their demonstrated ability for Cu(II) uptake and favorable structural characteristics, pelletized peanut hulls could gain use as a low-cost, once-through biomass filter medium for copper-bearing waste streams.

Synchronously pumped optical parametric oscillator driven by a femtosecond mode-locked fiber laser.

A femtosecond all-fiber laser source incorporating a cw mode-locked Yb-doped silica fiber oscillator and amplifier has been used to synchronously pump an optical parametric oscillator based on periodically poled lithium niobate. The signal output, consisting of 330-fs pulses at a 54-MHz repetition rate and average powers up to 90 mW, was tuned from 1.55 to 1.95microm , with a corresponding idler range of 2.30-3.31microm .

Extended operation of synchronously pumped optical parametric oscillators to longer idler wavelengths.

A synchronously pumped optical parametric oscillator, based on periodically poled lithium niobate, has operated at idler wavelengths out to 7.3 microm . Directly measured idler output powers (average) are 4.2 mW at 6.1 microm, 0.5 mW at 7.0 microm, and 0.04 mW at 7.25 microm . Characterization of the idler output indicates essentially bandwidth-limited and diffraction-limited operation.

Laser capture microdissection and real-time reverse transcriptase/ polymerase chain reaction of bronchiolar epithelium after bleomycin.

Terminal airways are affected in many lung diseases and toxic inhalations. To elucidate the changes in terminal airways in these diverse situations it will be helpful to profile and quantify gene expression in terminal bronchiolar epithelium. We used laser capture microdissection (LCM) to collect terminal bronchiolar epithelial cells from frozen sections of lungs of mice subjected to intratracheal bleomycin. The RNA from these cells was used for analysis of select messenger RNAs (mRNAs) by quantitative real-time polymerase chain reaction (PCR). In parallel, we used real-time PCR to analyze mRNAs in whole-lung homogenates prepared from other mice given intratracheal bleomycin. We found reductions of Clara cell-specific protein and keratinocyte growth factor receptor mRNAs in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchiolar epithelial transforming growth factor (TGF)-alpha mRNA was reduced but whole-lung TGF-alpha mRNA was not changed, whereas terminal bronchiolar epithelial epidermal growth factor (EGF) receptor mRNA was not changed but whole-lung EGF receptor was reduced. We conclude that LCM can isolate terminal bronchiolar epithelial cells for studies of cell-specific gene expression by quantitative real-time PCR, and that cell-specific gene expression in terminal bronchiolar epithelium is not necessarily reflected in analysis of whole-lung gene expression.