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Purpose: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal but clinically heterogeneous disease, with patients having variable benefit from endocrine and cytotoxic treatments. Intrapatient genomic heterogeneity could be a contributing factor to this clinical heterogeneity. Here, we used whole-genome sequencing (WGS) to investigate genomic heterogeneity in 21 previously treated CRPC metastases from 10 patients to investigate intrapatient molecular heterogeneity (IPMH).
Experimental Design: WGS was performed on topographically separate metastases from patients with advanced metastatic prostate cancer. IPMH of the RB1 gene was identified and further evaluated by FISH and IHC assays.
Results: WGS identified limited IPMH for putative driver events. However, heterogeneous genomic aberrations of RB1 were detected. We confirmed the presence of these RB1 somatic copy-number aberrations, initially identified by WGS, with FISH, and identified novel structural variants involving RB1 in 6 samples from 3 of these 10 patients (30%; 3/10). WGS uncovered a novel deleterious RB1 structural lesion constituted of an intragenic tandem duplication involving multiple exons and associating with protein loss. Using RB1 IHC in a large series of mCRPC biopsies, we identified heterogeneous expression in approximately 28% of mCRPCs.
Conclusions: mCRPCs have a high prevalence of RB1 genomic aberrations, with structural variants, including rearrangements, being common. Intrapatient genomic and expression heterogeneity favors RB1 aberrations as late, subclonal events that increase in prevalence due to treatment-selective pressures.
Translational Relevance
Intrapatient molecular heterogeneity in advanced metastatic castration resistant has been underexplored. Using whole-genome sequencing of 21 tumors from 10 patients, we show, in agreement with previous studies, an overall limited genomic heterogeneity in putative cancer drivers. However, private aberrations in putative drivers were identified. We also uncovered intrapatient heterogeneity in aberrations involving the RB1 gene and proceeded to study this in an independent cohort of metastatic castration-resistant prostate cancers. We show RB1 copy-number losses are common (∼56%) in tumors with previous exposure to taxanes and antiandrogens. We also identify genomic rearrangements as a common putative second hit. Finally, RB1 protein expression was heterogeneous in approximately 28% of tumors, which has implications for ongoing trials of CDK4/6 inhibitors, as the absence of functional RB1 is a negative predictive biomarker of response to these agents.
Prostate cancer is among the most frequently diagnosed malignancies worldwide (1). However, only about a quarter of all new cases will lead to cancer-specific death, highlighting the clinical heterogeneity of these tumors (1–3). Several large-scale genomic studies of cohorts of primary, hormone-naïve, and metastatic castration-resistant prostate cancers (mCRPC) have been performed suggesting both intra- and interpatient molecular heterogeneity (IPMH; refs. 4–11). Although multifocality and genomic heterogeneity are well-known features of primary prostate cancer (12–14), the level of IPMH in mCRPCs is less well established. Samples of late-stage disease are rarely acquired, with autopsy studies being expensive and logistically difficult, and few reports on IPMH in mCRPCs are available.
Two studies have investigated the extent of IPMH in mCRPC patients. In a comprehensive molecular study using array comparative genomic hybridization (aCGH), whole-exome sequencing (WGS), and transcriptional profiling, Kumar and colleagues reported that most presumptive driving genomic events and actionable targets are either present as common roots or "result from convergent evolution conferred by therapeutic pressures" (15). Further supporting this phenomenon of divergent genomics with convergent phenotypes, a WGS study of multiple metastases from 10 autopsied patients identified multiple separate endocrine treatment resistance mechanisms arising independently and subclonally within individual patients (16). Crucially, although both studies report some heterogeneity for known oncogenic drivers, neither followed up in dissecting gene-specific intrapatient heterogeneity using independent cohorts. Such efforts could shed light on the hierarchy of genomic events in prostate cancer progression.
The goal of this work was to identify recurrent intrapatient molecular diversity. We conducted WGS at >100X median coverage of 21 mCRPCs from 10 consecutive patients with >1 biopsiable nonbone metastases between January 01, 2013, to December 31, 2014. Our analyses, focused on 1,392 cancer-related genes, revealed intrapatient heterogeneity for the retinoblastoma-associated protein (RB1), a tumor suppressor that inhibits pro-oncogenic E2F1-mediated transcription in its hypophosphorylated state (17). In a study of 500 metastatic samples of 20 different tumor types, including 93 mCRPCs, RB1 was shown to be genomically aberrant in 13.6% of the cases, this being among the most common deleteriously aberrant genes in metastatic disease overall (18). Critically, aberrations of RB1 are commoner in mCRPC, indicating that these are either present at onset in clinically aggressive primary tumors or are acquired late in the natural history of prostatic malignancies.
We then employed FISH and IHC to further study RB1 loss and show that RB1 genomic aberrations are common in clinically aggressive primary prostate cancer, becoming commoner in mCRPC regardless of histology. We demonstrate that structural variants involving RB1 are frequent in mCRPCs and may be a common putative second hit. Finally, we show that heterogeneous RB1 protein loss is common, favoring RB1 depletion as a late event in the natural history of prostate cancer.
Materials and Methods
Population and tissue samples
Patients gave their written-informed consent and were enrolled to protocols approved by the Royal Marsden NHS Foundation Trust Hospital (London, UK) ethics review committee (reference no. 04/Q0801/60). Demographics and clinical data were retrospectively collected from electronic hospital records. WGS data were generated from 21 samples from 10 consecutive patients with >1 accessible nonbone metastases acquired between January 01, 2013, and December 31, 2014. In addition, FISH and IHC data were generated from randomly selected metastatic samples collected between January 01, 2013, and December 31, 2014. In this second cohort, metastatic sites included lymph node, bone, viscera, and soft tissues. Minimum tissue quality criteria required the presence of at least 50 well-preserved cancer cells. Patient-matched hormone-sensitive tumors for a subset of cases were studied.
For each of the 21 metastases, tumor DNA was extracted from seven 10-μm-thick frozen sections after confirmation of tumor cell presence on a 4-μm-thick hematoxylin and eosin (H&E)–stained slide. Germline DNA was extracted from saliva. The QIAmp DNA Mini Kit (#51304; Qiagen) was used for DNA isolation from both fresh/frozen sections and buccal swabs. Library preparation was performed using the TruSeq Nano DNA Library Prep Kit. Sequencing was performed using the Illumina HiSeq 2000 System. Tumor and matched germline DNA samples were profiled with a WGS protocol. Paired-end sequencing reads were aligned to the human reference genome (GRCh37/hg19) using Illumina Isaac Genome Alignment Software (19) embedded in Illumina pipeline version v2.0.1. Genome Analysis Toolkit (GATK v2.6; ref. 20) best practices for variant calling that included marking of duplicate reads, recalibration of base quality scores, and local realignment were adopted for all aligned samples. Germline–tumor pair consistency was tested using the SNP panel identification assay (SPIA v1.1.0; ref. 21). To identify and characterize somatic single-nucleotide variants (SNV) in exons, the MuTect v1.1.6 (22) tool was used. To reduce false positives, calls were refined and confirmed using an ad hoc pileup approach using ASEQ v.1.1.8 (23) and adopting stringent filtering quality criteria. Finally, each retained SNV was annotated with genomic features and effect/impact prediction using the SnpEff v4.1a tool (24). For each tumor and matched normal WGS sample, the algorithm BreakDancer v1.2 (25) was run with default parameters to detect genomic SVs including intra- and interchromosomal translocations. The raw output of BreakDancer was filtered using stringent criteria based on quality score and minimum number of reads supporting the SV. In addition, an ad hoc WES-based computational strategy was developed to support evidence of significant enrichment in the coverage of exons involved in tandem duplications detected in the RB1 gene. Briefly, the mean coverage in each RB1 exon was computed separately in tumor and normal samples and normalized to the total coverage of all RB1 exons (N = 27). Then, the ratio between tumor and matched normal normalized mean coverages was computed for each RB1 exon. Finally, the method computed the median ratio grouping exons 1 to 6 (Rupstream), 7 to 17 (Rtandem duplication), and 8 to 27 (Rdownstream). Tandem duplication events were compatible with high ratios Rtandem duplication/Rupstream and Rtandem duplication/Rdownstream. Estimation of genomic segments and somatic copy-number aberrations (SCNA) was obtained using BICseq v1.1.11 algorithm (26). Segmented data were used in combination with CLONET v2.0.0 (27) to estimate ploidy and purity for each tumor sample and further to determine allele-specific copy number.
Cell lines
22Rv1 (ATCCCRL-2505), a prostate cancer cell line, and MD-MBA-468 (ATCCHTB-132), a triple-negative breast cancer cell line, were purchased from the ATCC and cultured according to the manufacturer's protocol. Cell pellets were fixed in 10% neutral buffered formalin overnight at 4°C. After dehydration in ethanol, at 70% (10 minutes), 80% (10 minutes), 90% (10 minutes), and 100% (3 × 10 minutes), lipid removal and diaphonization were performed with xylene (3 × 15 minutes).
Antibody validation, IHC, and scoring methods
Antibody target specificity validation was confirmed by performing Western blots of cell lysates as previously described (28). For IHC, formalin-fixed paraffin-embedded tissue samples were cut at 4-μm-thick sections onto glass slides and dewaxed with xylene. Antigen unmasking was performed by heating slides in a pH6 citrate buffer solution for 18 minutes using a microwave at 900 W. Staining was performed with conventional diaminobenzidine method using the i6000 autostainer (Biogenex). The Dako-Envision kit (Agilent-Dako) was used for reaction visualization. A mouse monoclonal anti-RB1 antibody targeting amino acids 332–344 (Clone G3-245, BD Biosciences) diluted 1:50 was used. Endogenous peroxide was blocked using a 3% H2O2 solution; nonspecific staining was blocked using Dako protein block serum-free X0909, and the diluted primary antibody was incubated on tissue samples for 1 hour. We used MDA-MB-468, a triple-negative breast cancer cell line, with biallelic deletion of the RB1 locus (c.265_2787del2523) as negative control and 22RV1, an AR-positive prostate cancer cell line with genomically intact RB1 as a positive control. Absence of IHC AR staining (Clone AR441, Agilent-Dako) was used as a surrogate marker of neuroendocrine prostate cancer (NEPC). This validated anti-AR antibody assay was performed as previously described (28). An H-Score determined by the formula, (% of negative) × 0 + (% of weak positivity) × 1 + (% of moderate positivity) × 2 + (% of strong positivity) × 3, yielding a result between 0 and 300, was used for RB1 protein expression semiquantitative analyses. Samples with any proportion of tumor cells lacking RB1 staining with good internal controls, i.e., RB1-positive endothelial or stromal cells, were interpreted as heterogeneous, and positivity was considered of weak intensity to reflect an H-Score of <100. AR was dichotomously scored as either positive or negative, with the cutoff for positivity being expression >0.
FISH was performed as previously described (29). We optimized a dual-color FISH assay using two commercially available FISH probes: (1) an approximately 202 Kb probe directly labeled with Spectrum Orange targeting the 13q14 locus spanning RB1, and (2) an approximately 612 Kb probe directly labeled with Spectrum Green targeting the subtelomeric 13q34 locus (Abbot Laboratories). A subtelomeric control probe was selected because homologies in centromeric alpha satellites between chromosomes 13 and 17 could compromise gene copy-number enumeration (30). We defined copy-number gains as a ratio of RB1 signals/nuclei ratio of >2; copy-number neutral (CNN) as a ratio of RB1 signals/nuclei ratio of <2 and ≥1.7; shallow deletions (SD), i.e., mostly heterozygously deleted, as a ratio of RB1 signals/nuclei <1.7 and ≥1; and deep deletions (DD), i.e., at least focally homozygously deleted, as a ratio of <1. At least 50 intact nonoverlapping nuclei were counted per sample, and the number of cells with >2, 2, 1, or 0 signals was recorded for both probes. The controls used for RB1 IHC were the same as those used for FISH, namely MDA-MB-468 as negative and 22RV1 as positive controls, respectively.
Figures and statistical analysis
Figures were generated using R version 3.3.2 and GraphPad Prism v7. Statistical tests were performed using GraphPad Prism v7 and STATA v15.
Ethics statement
The work herein presented was conducted to Good Clinical Practice standards and in accordance to the Declaration of Helsinki.
Clinical features of the patients selected for WGS
Patients in this study had clinically aggressive disease, the majority being stage IV at diagnosis; their clinical data are summarized in Table 1. Median age of the WGS cohort was 58.8 years [inter-quartile range (IQR): 50.84-61.4]. Gleason scores were available for 9 patients, 4 having scores of ≤7 and 5 of >7. Two patients had local treatment with radical prostatectomy followed by salvage radiotherapy. Another 2 patients had radical radiotherapy. Eight of the 10 patients had locally advanced disease, i.e., ≥T3, with 6 having M1 disease, at diagnosis. Median time on androgen deprivation therapy was 14.99 months (IQR: 6.95–49.15 months). A summary of all patient treatments and best clinical responses are available in Supplementary Fig. S1. Twenty-one (21) mCRPC biopsies were collected from these 10 patients; 2 samples per patient except one subject from whom we had three separate nodules from a penectomy specimen. In 7 patients, these mCRPC samples came from enlarged, topographically distant, lymph nodes; in two men, liver mCRPC biopsies and lymph-node biopsies were available; in one subject liver and muscle mCRPC biopsies were acquired. No complications occurred during tumor sampling. All biopsies included a fresh-frozen sample for genomic sequencing studies and a formalin-fixed, paraffin-embedded sample to confirm tumor histology and for IHC studies. Fresh-frozen samples were cut at 4 μm and rapidly stained with H&E to confirm tumor content. Nineteen of 21 samples had >50% tumor content, with the remaining two cases having approximately 10% and 25%.
Bulk tissue WGS shows limited intrapatient genomic heterogeneity
We obtained WGS data from these 21 tumor samples from 10 patients; 21 tumor and 10 matched germline DNA samples were profiled at a median sequencing coverage depth of 109X and 37X, respectively. Genomic profiles of tumor samples were defined from WGS data by applying an ad hoc computational workflow. CLONET estimated tumor ploidy, purity, and corrected SCNA in all but 2 tumor samples that showed low quality data. Overall, tumor cellularity was high (median = 74%, min = 38%, max = 94%), and polyploidy (genomes with more than 2 paired sets of chromosomes) was detected in all tumor samples.
Our initial approach focused on genes of taxonomical relevance, i.e., clonal, mutually exclusive events that define genomic subtypes (31) and previously reported by the SU2C International team CRPC study (11). We identified ETS rearrangements in 13 samples from 6 patients; in 11 of these samples from 5 patients, ERG was the ETS partner involved. WGS-segmented data indicated the presence of an interstitial deletion spanning approximately 3 Mb between ERG and TMPRSS2 in 7 samples from 3 patients (V4074, V5162, and V4038). Breakpoints of this deletion were consistent among samples within the same patient. In 2 samples from 1 patient (V4002), we identified a previously unreported fusion between ETV1 and MIOS. ETS rearrangement statuses were identical in different samples from an individual patient. The remaining 4 ETS rearrangement–negative cases did not have clonal somatic mutations in SPOP, IDH1, or FOXA1, subgroup-defining genomic lesions described by The Cancer Genome Atlas (TCGA) consortium (4).
The landscape of identified coding variants of interest through this WGS is summarized in Fig. 1. First, we quantified the total number of somatic single-nucleotide variants (SNV) in coding regions for each tumor sample and found a median number of 173 (min = 127, max = 494) variants. When the analysis was stratified by functional annotation, the median number of nonsynonymous SNVs was 43 (min = 27, max = 127). The two tumors harboring the highest number of SNVs, with 494 and 456 bases mutated (127/494 and 123/456 being nonsynonymous SNVs), were from the same patient and presented a deleterious mismatch repair gene aberration (i.e., missense variant p.Ile185Val in MLH3). Second, we estimated for each sample the fraction of the genome that was affected by a SCNA, here defined as copy-number altered fraction (CNAF). The median CNAF across tumor samples was 0.83 (min = 0.35, max = 0.94). These fractions were significantly higher than those reported by Hieronymus and colleagues in aCGH and low-pass WGS studies in hormone-sensitive primary and metastatic prostate cancers, with CNAF of 0.04–0.05 and 0.32, respectively (32). Our data indicate that CNAF increases significantly in later-stage mCRPC, suggesting that increased aneuploidy favors cell fitness and treatment resistance. To quantify the somatic CN similarities among metastatic sites from the same patient, we measured the correlation between log2 ratios in a set of 1,392 cancer-related genes. Results based on log2 ratios of these genes indicated overall strong correlations with a median of 0.85 (min = 0.53, max = 0.95). We also focused on a subset of genes (N = 32), reported in Fig. 1 and selected based on their relevance to prostate carcinogenesis and key roles in important molecular pathways. The fraction of these genes with concordant copy-number status across metastases from the same patient was on average 0.93 (median = 0.95, min = 0.75, max = 1). Overall, our somatic SNVs and CN analyses to assess intrapatient heterogeneity confirmed low-level diversity at a bulk tumor level as previously reported (15).
However, genomic heterogeneity was identified in some patients. Aberrations in the WNT signaling pathway were seen as private events in two metastases from 2 distinct patients; the first showing a missense point mutation causing the protein change Ser680Arg in CTNNB1, with the latter showing a missense variant in APC (protein change Gly108Val). Amplification of AURKA, a serine-threonine kinase with key roles in centrosome maturation and spindle assembly (33), was seen in one of two samples from a single patient (V4038). Finally, in 2 patients (20%; 2/10), heterogeneous somatic genomic aberrations of RB1 were identified between different metastases (Fig. 1). In the first patient (V5128), one metastatic site had CNN LOH with the other site having heterozygous loss. A second patient (V5033) had heterozygous loss in both metastases, but an additional single-base substitution causing premature protein truncation at Glu204 in only one of them. Importantly, CLONET-based tumor purity estimations of these samples were high (94%, 74% and 85%, 84%).
RB1 copy-number enumeration by FISH and protein expression in mCRPC
Given the observed intrapatient heterogeneity of RB1 loss in mCRPC, and the fact that these aberrations were more common than anticipated in mCRPC samples, we decided to further investigate RB1 loss by orthogonal assays pursuing RB1 FISH and RB1 IHC. We hypothesized that the presence of RB1 deletions in mCRPC from patients with matched, previously copy number normal, hormone sensitive prostate cancer (HSPC) samples, as well as the presence of heterogeneous IHC staining in mCRPC biopsies would both favor disruptions of RB1 as late, subclonal, molecular events. These would have therapeutic relevance because RB1 loss of function is a putative predictive biomarker of resistance to CDK4/6 inhibitors (34), which have antitumor activity in ER+/HER2− breast cancer (35, 36) and are being evaluated in clinical trials of mCRPC (NCT02905318). The validation of RB1 FISH and IHC assays is summarized in Supplementary Fig. S2. The MDA-MB-468 cell line had complete absence of IHC staining with rare cells having a single RB1 FISH signal, resulting in an RB1 signal to nuclei ratio (RB1s/Nuc) of 0.43 in 100 nuclei counted. The 22RV1 cell line had diffuse RB1 positivity on IHC and an RB1s/Nuc ratio of 2.03 in 100 nuclei counted. To validate copy-number analyses determined by sequencing and to query RB1 heterogeneity at the protein level, we analyzed the 21 samples from 10 patients in the WGS cohort using RB1 IHC and FISH. FISH results were successfully acquired from 20 of 21 samples, with 19 of these also having RB1 copy number determined by WGS; FISH copy-number analyses were consistent with WGS estimates in 16 samples (84%). All seven cases identified as hemizygously deleted for RB1 by WGS also had RB1 signal/nucleus ratios of <1.7 by FISH enumeration, which validated our cutoff to define RB1 loss. Interestingly, one case was considered RB1 wild-type (WT) by WGS but had an RB1/nucleus FISH ratio of 1.01, whereas two cases had ratios >2 but were reported WT by WGS. These discrepancies may have resulted from stromal contamination or pseudonormalization of genomic data in polyploid tumors. A comparison between RB1 copy-number evaluation by WGS and FISH, RB1 IHC, and histologic tumor type is presented in Supplementary Table S1.
Structural variants of RB1: a putative mechanism of RB1 inactivation
The identification of four mCRPCs with complete absence of RB1 expression without a significant proportion of cells showing genomic copy loss led us to hypothesize that other genomic events could account for a presumptive inactivation mechanism. Exploiting the WGS data, we identified four distinct putative SVs involving RB1 in 6 metastases (6/21; 28.57%) from 3 patients (3/10; 30%); 5 of these 6 mCRPC biopsies (83.33%) had complete protein loss by IHC. Three of the four SVs were interchromosomal translocations, and one was an intrachromosomal tandem duplication. In 2 of these patients, these SVs were shared by multiple metastatic samples (S115, S116, and S117 in patient V4074; and S363 and S364 in patient V5191; all of these samples were IHC negative). In the third patient (V4038), a single sample presented with two distinct RB1 SVs but preserved IHC expression.
In three samples with ADC histology from patient V4074, our computational analysis revealed RB1 CNN LOH profile. Concomitantly, an increment in the number of sequencing reads spanning exons from 7 to 17 suggested an intragenic tandem duplication of the genomic segment containing exons 7 to 17. Using matched RNAseq data, we were able to validate the abnormal transcript generated by this intragenic tandem duplication that would impair protein translation. Next, we first queried WGS data from the same patient and verified the capability to detect the genomic event by such a strategy, and next queried a large set of individuals (11) for the same event (N = 149; Fig. 2). Although this specific RB1 structural event associating with full protein loss does not appear to be frequent, it evidences the importance of appropriate assay design to accurately query RB1 status for patient management.
RB1 deletions are frequent in clinically aggressive HSPC and more common in mCRPC
To further evaluate RB1 loss heterogeneity in lethal prostate cancer, we generated FISH data for RB1 copy-number analysis from 70 samples from 41 patients (including the 19 samples with WGS copy-number data available). Importantly, the set included 20 HSPC and 50 CRPC biopsy samples, including 20 matched, same-patient HSPC–CRPC pairs (Fig. 3A). Of the 20 HSPC samples, 35% (7/20) had at least shallow RB1 deletions, i.e., a gene signal/nuclei ratio <1.7, compared with 65% (13/20) of the matched, same patient, CRPC samples. Of the 13 HSPC samples with ratios ≥1.7 prior to any systemic therapy, 7 had matching CRPC samples with ratios <1.7, whereas 6 retained a ratio ≥1.7. Of the 7 HSPC samples with ratios <1.7 at diagnosis, only one matched CRPC sample had a ratio ≥1.7. Our data indicate an enrichment for RB1 deletions in clinically aggressive HSPCs, in line with a previous study on nonindolent prostate cancer (37). More importantly, however, we also show RB1 deletions evolving in matched mCRPC from previously CNN HSPCs.
Deletions of RB1 in mCRPCs
Overall, of the 50 mCRPC samples with FISH data, 43 were adenocarcinomas, 6 were NEPCs (cohort was enriched for NEPC), and 1 had mixed histology with predominantly (∼95%) NEPC phenotype. An XY plot showing ratio between RB1/nuclei versus IHC H-Score and histologic type notation is seen in Fig. 3B. Copy-number estimation of RB1 by FISH revealed a high degree of heterogeneity, with most tumors showing a subset of RB1-deleted cells larger than could be attributed by artifactual nuclear truncation, i.e., tangential sectioning of nuclei. Only 12 (24%; 12/50) mCRPCs were CNN for both RB1 and reference probe. RB1 deletions, shallow or deep, were evident by FISH in 56% (28/50) of the mCRPC samples; SDs were noted in 10 samples and DDs in 18 samples. The reference probe was not helpful for RB1 copy-number enumeration as it was detected in various combinations being detected to be lost, neutral, or gained independently of RB1 status, indicating frequent structural aberrations involving the 13q34 locus.
Relationship of RB1 FISH enumeration and RB1 protein expression by IHC
Twenty-two of 43 ADCs (22/43; 51.16%) had diffuse RB1 IHC positivity (IHCPos; Fig. 3B); RB1/nuclei FISH ratios for these 22 tumors was >2 in 5, <2 and ≥1.7 in 8, and <1.7 in 9. Critically none of the diffusely positive cases had an RB1/nuclei ratio of <1. In the 16 ADCs with IHC heterogeneity (IHCHet), RB1/nuclei FISH ratios were >2 in 1, <2 and ≥1.7 in 2, and <1.7 in 13, of which 5 were <1. Of the 5 ADC with RB1 IHC–negative staining (IHCNeg), FISH profiles showed 4 cases with an RB1/nuclei ratio <2 and ≥1.7, and one with a ratio of 1.16. Of the seven NEPC samples, one had a ratio >2, one <2 and ≥1.7, and 5 had ratios <1.7, of which 3 were <1. All seven NEPCs with available FISH data were RB1 IHCNeg.
Kendall's Correlation Coefficient was used to determine the association between RB1 signal/nuclei ratio and H-score. Tumors with an RB1s/nuclei ratio <1.7 were defined as having loss; ROC curves with bootstrapped confidence intervals were used to determine an optimal cutoff for RB1 H-score to detect cases with a FISH ratio of ≥1.7. The area under the ROC curve was 0.55 (95% confidence interval, 0.39–0.74) with an optimal cut-point for H-score of 105 (sensitivity, 0.77; specificity, 0.25; Supplementary Fig. S3). Overall, these data indicated that RB1 copy-number losses are common in mCRPCs but did not correlate well with RB1 IHC protein loss of expression. As demonstrated in the WGS cohort, this was likely due to undetected complex structural rearrangements as putative second hits to RB1.
To better understand the relationship between RB1 genomics and protein, we turned to the IHC and WGS data of the 21 tumor samples to pursue a detailed characterization of RB1 loss. For each sample, we annotated genomic aberrations with potentially damaging functional impact, such as copy-number loss, somatic missense SNVs, and structural rearrangements (tandem duplication and chromosomal translocations) affecting the coding region of RB1. These studies showed that RB1 protein expression by IHC correlated well with the total number of genomic aberrations detected; the higher the number of genomic aberrations at the RB1 locus, the lower the mean RB1 protein level. Specifically, tumors carrying no (N = 7), one (N = 8), or two (N = 6) genomic aberrations in RB1 had median IHC scores of 120 (mean = 120, SD = 35.11), 65 (mean = 61.25, SD = 57.92), and 0 (mean = 0, SD = 0), respectively (Fig. 4).
RB1 expression can be highly heterogeneous in advanced prostate cancer
Different RB1 IHC staining patterns were observed in mCRPC biopsies (Table 2). Overall, homogenous RB1 IHC staining (positive or negative) was seen in 71.7% of the samples (76/106). Of these, 56.6% (60/106) of mCRPC biopsies were positive and 15.1% (16/106) negative (Fig. 5A1-4 and 5B1-4). When categorizing RB1 IHC expression by histologic phenotype, 61.4% (59/96) of ADCs and one NE cancer (12.5%; 1/8) showed homogeneous positivity (Fig. 5A4). Complete loss of RB1 expression was seen in 8.3% (8/96) of ADCs (Fig. 5B1-2) and in 87.5% (7/8) of NE tumors (Fig. 5B3-4). Two cases showed mixed histology with coexistence of adenocarcinoma and AR-low NEPC. One of these cases had homogenous loss of RB1 IHC expression in both components, whereas the other showed positivity in the ADC component and negativity in the NE component in a single bone metastases biopsy (Supplementary Figs. S5 and S6). Heterogeneous RB1 staining patterns were noted in 28% (30/106) of mCRPC samples (see Fig. 5C1-4), with most of these showing an ADC phenotype (96.6%; 29/30) apart from one sample which had mixed histology (Fig. 5C4; Supplementary Fig. S6). Our data indicate that heterogeneous/focal RB1 protein loss of expression is common in mCRPC corroborating reports of loss of RB1 as a late event and associated with treatment selective pressures.
CRPC is invariably lethal. However, substantial interpatient genomic heterogeneity and variable sensitivity to established treatments, including endocrine agents and taxane chemotherapy, lead to variable clinical courses. Robinson and colleagues suggest that "nearly 90% of cases have potentially actionable somatic or germline events" (11). Molecular stratification of mCRPC promises to improve the treatment of these diseases (38, 39), but these strategies are challenged by intrapatient heterogeneity, which can be difficult to identify from bulk exome sequencing. Intrapatient heterogeneity results in mixed responses to anticancer drugs; imaging studies, for example, have shown divergent interlesion responses to the PARP inhibitor olaparib in individual patients with advanced solid tumors (40). Identifying significant intrapatient genomic heterogeneity in mCRPC is thus key to validating biomarkers and deliver molecularly stratified treatments.
Herein, we obtained WGS of mCRPC tumor biopsies at 100X mean coverage and performed focused analyses on somatic variants involving 1,392 cancer-related genes. In agreement with the data reported by Kumar and colleagues (15), despite significant interpatient heterogeneity, the majority of somatic genomic aberrations were shared between different biopsies of each individual patient at the bulk level. Events involving taxonomy defining genes, e.g., ETS fusions, and certain recurrently aberrant tumor suppressors such as TP53 mutations and deletions of PTEN, were consistently shared between samples from the same patient, in keeping with these being earlier, i.e., prior to CRPC state, events (31, 41, 42). Certain cancer-related genes previously reported in prostate cancer, however, were identified as private events, favoring later, likely subclonal origins. These included AURKA amplification, mutations in WNT-signaling genes, and RB1 aberrations. Because TCGA and SU2C/PCF sequencing data suggested an increase in RB1 loss in mCRPC compared with primary disease and because of the established role for RB1 loss as a predictive biomarker of CDK4/6 inhibitor resistance (34), we pursued a deeper study of RB1 in a wider cohort of mCRPC biopsies. We found that intrapatient heterogeneity for RB1 loss is common in mCRPC both at the genomic and protein levels. Highly relevant for the current treatment of mCRPC, we uncover novel complex genomic RB1 structural variants that affect RB1 protein expression and that would have gone undetected by exon-focused sequencing assays. This discovery supports the use of IHC, and perhaps multiple orthogonal assays, for determining evidence of RB1 loss.
In mCRPC, RB1 inactivation can promote survival benefit via distinct mechanisms including an increased output of AR-directed transcription (43) or, in combination with inactivation of TP53, by inducing a state of plasticity which allows the emergence of AR-low NEPCs (44–46). Using WGS, Fraser and colleagues identified RB1 losses, mono- or biallelic, in 35% of nonindolent HSPCs (37), a similar frequency to what we observed in the HSPC samples from our mCRPC-matched, same-patient, cohort. Furthermore, in agreement with previous studies (5, 9, 11), we identify a higher frequency of RB1 loss in mCRPC (56%) than in HSPC (35%). This is further evidence that RB1 aberrations, including monoallelic deletions, confer fitness advantages to prostate cancer cells. Critically, in agreement with our data, a study evaluating SCNAs from WGS data from HSPCs and mCRPC found that partial losses of RB1 are significantly more common than homozygous deletions (47). We identified intrasample heterogeneous loss of RB1 expression by IHC in 28% of our mCRPC samples, implicating RB1 depletion as a late and subclonal event. We also observed an erratic relationship between RB1 IHC protein loss and copy-number status as determined by FISH. Samples with RB1 monoallelic deletions showed both homogeneous and heterogeneous expression patterns by IHC, and some CNN cases had complete absence of protein expression. These data indicate that CN status alone, assessed by either massive-parallel sequencing of bulk biopsies or FISH, provides insufficient information on RB1 protein expression and function. Even when protein expression is present, however, determining RB1 functionality is challenging. Using mRNA-derived signatures from bulk sequencing has been a recurrent approach (15, 43), but focal RB1 loss of expression/loss of function may become diluted in tumors with large proportions of RB1 functional components.
Our study has limitations. Firstly, our patient cohorts were retrospectively collected and heterogeneous in terms of prior treatments with different timing of biopsies in relation to treatment lines. This impairs our ability to draw meaningful clinical conclusions regarding the prognostic value of the biomarkers we studied. Secondly, we were unable to obtain satisfactory RB1 staining from diagnostic HSPC samples. This could be due to different factors including tissue block age (several blocks were over a decade old) and the fact that these samples were acquired from multiple pathology laboratories with different tissue processing protocols. In contrast, our mCRPC samples were, at maximum, up to 3 years old and were uniformly processed to rigorous protocols. Finally, we did not perform assays that could inform on epigenetic silencing of RB1 and thus are unable to assess the contribution of methylation events to RB1 inactivation.
In conclusion, previous studies indicate that the proportion of advanced prostate cancers showing 13q14 (locus of RB1) mono- or biallelic deletions ranges from as low as 10% (11) to as high as >90% (8) depending on the assay used for gene copy-number estimation. Herein, we report a frequency of RB1 loss by FISH of 56% and intrasample focal IHC losses of RB1 expression in 28% of mCRPCs. Our findings support RB1 aberrations in mCRPCs as resulting from treatment selection pressures in most cases. These data are critically important to the study of CDK4/6 inhibitors in mCRPC, with such trials now ongoing (e.g., NCT02905318). Overall, our data indicate that a significant number of mCRPC have heterogeneous loss of RB1 protein suggesting CDK4/6 inhibitors may be less efficacious in later-stage mCRPC with efficacy being limited to patients without RB1 depletion.
Disclosure of Potential Conflicts of Interest
J. Mateo reports receiving speakers bureau honoraria from Sanofi. J.S. de Bono reports receiving speakers bureau honoraria from Astellas, AstraZeneca, Daiichi, Genentech, Genmab, GlaxoSmithKline, Janssen, and Pfizer. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: D. Nava Rodrigues, G. Boysen, F. Demichelis, J.S. de Bono
Development of methodology: D. Nava Rodrigues, M. Crespo, S. Miranda, I. Figueiredo, F. Demichelis, J.S. de Bono
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Nava Rodrigues, M. Crespo, S. Miranda, P. Rescigno, R. Riisnaes, S. Carreira, S. Sumanasuriya, J. Mateo, F. Demichelis, J.S. de Bono
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Nava Rodrigues, N. Casiraghi, A. Romanel, P. Gasperini, J. Mateo, A. Makay, K. Knudsen, G. Boysen, F. Demichelis, J.S. de Bono
Writing, review, and/or revision of the manuscript: D. Nava Rodrigues, N. Casiraghi, A. Romanel, M. Crespo, S. Miranda, P. Rescigno, I. Figueiredo, R. Riisnaes, S. Carreira, S. Sumanasuriya, A. Sharp, J. Mateo, C. McNair, M. Schiewer, K. Knudsen, G. Boysen, F. Demichelis, J.S. de Bono
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Sumanasuriya, J.S. de Bono
Study supervision: F. Demichelis, J.S. de Bono
We would like to acknowledge funding and support from the European Research Council Consolidator Grant 648670 (F. Demichelis), Movember, Prostate Cancer UK, Prostate Cancer Foundation/SU2C to the International Prostate Cancer Dream team, Cancer Research UK, Experimental Cancer Medicine Centre grant funding from Cancer Research UK and the Department of Health, and Biomedical Research Centre funding to the Royal Marsden. We would also like to thank Dr. Scott Tomlinss and Dr. Angelo De Marzo for their advice on antibody selection and assay development. This research was also supported by a Stand Up To Cancer-Prostate Cancer Dream Team Translational Research Grant (SU2C-AACR-DT0712). Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
- Received June 29, 2018.
- Revision received August 9, 2018.
- Accepted September 18, 2018.
- Published first September 26, 2018.
- ©2018 American Association for Cancer Research.


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40th Year Celebration & Ribbon Cutting
The Chamber will also be holding a ribbon cutting for Leyco under new ownership - Currie Luedecke & Kim Johnson.
Everyone is invited to join in the festivities.
Name: 40th Year Celebration & Ribbon Cutting
Date: November 16, 2018
Time: 12:00 PM - 2:00 PM CST
Website: http://www.leycorealestate.com
Event Description:
Leyco Real Estate is 40 years young and they are celebrating!

subset: commoncrawl


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In July 1956, the SS Andrea Doria sank on the last night of its voyage from Italy to New York City. With over 1,134 passengers aboard, including Hollywood movie stars Ruth Roman and Betsy Drake, barely 100 short of her passenger capacity of 1,241. There were 190 first class passengers, 267 cabin class passengers and 677 tourist class passengers, which ... Read More »
Clive Palmer's Titanic II pitch fails to wet the sails…
Billionaire Clive Palmer's pitch about Titanic II – the ship he is funding to be built before a maiden 2020 voyage – hardly had the imagined grandeur and prestige of the original ship's build in the early 1900s. The mining magnate and former federal Australian MP featured in a bizarre promotional video last week for Blue Star Line, the company ... Read More »
Home Line's OCEANIC was one of the most beautiful and successful cruise ships of all times.
By the mid-1960s, when jets had killed off all but the stragglers on the North Atlantic, Home Lines introduced the OCEANIC. Home Lines proudly proclaimed that their 39,000 tonner was "the largest ship built exclusively for cruising." By the mid-1960s, when jets had killed off all but the stragglers on the North Atlantic, Home Lines introduced the OCEANIC. Once it ... Read More »
The luxurious liner Awatea was the "only way to cross" the Tasman Sea from Australia to New Zealand!
Far away from the Trans-Atlantic services – "Down Under" – Union Steam Ship Company operated a fleet of excellent passenger ships between Australia and New Zealand until 1960. In her day the Awatea was regarded as one of the most luxurious and fastest liners of the period. Her history was brief and she was destroyed in World War2 like so ... Read More »
RETRO look at Mid-Century ship travel and cruising in the 1950s and 1960s
Travel by cruise ship or passenger liner in the 1950s and 1960s was far different from today's cruising. Many passengers were traveling from one point to another, they had a destination, while others were taking cruises. You could be sailing across the pond to Europe for business or pleasure, heading out of New York to the West Indies or South ... Read More »
TSS Awatea – 1930s crossing between Australia and New Zealand.
The TSS Awatea was one of the most beautifully designed compact liners to be built in the mid-thirties and was, without doubt, the ultimate statement in luxurious service. The Awatea followed the Art Deco trend of the day. The interiors were distinguished by fluorescent lighting, aluminum motifs, and gentle pastels throughout the ship that created an understated elegance. She was ... Read More »
SS LIBERTE – Crossing the Atlantic with Elizabeth Taylor and all the stars!
As the SS LIBERTE, the liner became the stop gap flagship for CGT French Line as a replacement for the legendary SS NORMANDIE that had been lost during the Second World War. If the time before the Second World War is indexed within the annals of history for its aspirations of achievement and advancement of technology and design, then, the ... Read More »
Photos of the RMS TITANIC rescue and the "Unsinkable" Molly Brown sold at auction for US 42 K
The album belonged to Louis M Ogden, a New York socialite aboard the RMS Carpathia – the ship that rescued survivors of the Titanic. Carpathia passenger's scrapbook of the Titanic rescue – including a photo of the Unsinkable Molly Brown's lifeboat New York socialite Louis M. Ogden's scrapbook includes several pictures showing the rescue of survivors of the Titanic sinking. Ogden ... Read More »
On board the Ward Line's SS MORRO CASTLE, in the early morning hours of September 7, 1934, a deadly fire erupted in two places on the cruise/liner en route from Havana Cuba to New York City. After wonderful days at sea and a wonderful time in Havana, the cruise turned into a nightmare. Nothing could have been more horrendous for ... Read More »
History of Cruising – From the Britannia to the Love Boat
EARLY CRUISING The earliest ocean-going vessels were not primarily concerned with passengers, but rather with the cargo that they could carry. Black Ball Line in New York, in 1818, was the first shipping company to offer regularly scheduled service from the United States to England and to be concerned with the comfort of their passengers. TRANSATLANTIC BY SAIL and STEAM ... Read More »
Mid-Century Modern – The Delta Line sails to South America
The Delta Line was cruising to South America from New Orleans in the 1950s. The steamship company introduced three revolutionary passenger-cargo ships to its South American services in the post-war years of the 1940s. In keeping with the trade name of the company, "Delta Line," the three vessels were given "Del" names DEL NORTE, DEL SUD, and DEL MAR. The ... Read More »


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Derma-Rx Advanced Skin Renewal Serum
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Call 6733 1555 (Tong Building Singapore Branch) / 6822 7035 (Wisma Atria Singapore Branch) / 603 2284 1088 (Mid Valley Malaysia Branch) for an appointment.
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AC Milan forward M'Baye Niang has attracted the interest of various clubs around Europe, according to his agent.
West Ham United, Everton, Leicester City have been linked with former Caen forward in recent months, since Milan's dismal showing in 2015/16 Serie A campaign – finishing in seventh.
Moussa N'Diaye, who represents the young Frenchman, claims there have been enquiries about his availability from English Premier League and Bundesliga sides.
"Talk of formal offers for him is premature," he told Italian website Footmercato.
"There is interest, but no actual proposals yet. As happens in every transfer session, he is linked with many clubs.
"His potential emerged after the experience on loan at Genoa. His style of football is very interesting.
"There are several options, including in England and Germany, but no rush."


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Install Cardcaptor Sakura New Tab Theme and enjoy handpicked HD images of Cardcaptor Sakura.
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If you liked the Cardcaptor Sakura extension we suggest you to take a look our other extensions. You can reach these extension by visiting www.choosetab.com


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Cone Crusher - Sep 26, 2016 . Cone Crusher . Hydraulic retraction system for ease of movement between transport and . Hydraulic overload protection with automatic reset.cone crusher hydraulic protection system,sbs cone crushers - One simple power unit in a SBS series cone crusher is used to power all its hydraulic systems: • Hydraulic Overload Protection. • Dynamic Adjustment.
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In the Kubria® Cone Crusher the material is crushed by compressive forces between the . system especially designed for thyssenkrupp Cone Crushers. . Hydraulic main shaft adjustment . Protection system for automatic tramp release.
TRIO cone crusher's use evenly positioned hydraulic cylinders for thread lock, which . TRIO also utilizes an innovative sealing system to isolate dust and protect.
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Apr 4, 2017 . Whether you order a complete system, . Our Trio® TC series cone crushers are ideal for heavy duty secondary .. Hydraulic system protection.
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At the heart of the Maxtrak is the Automax® cone crusher with hydraulic setting, tramp release . Cone overload protection. ▫. Metal detector. ▫. Dust suppression system. ▫. Economical to operate with a highly fuel efficient direct drive system.
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Started as an ill-advised solo project mashing fuzzy guitar rock, orchestral instrumentation and the sounds of 80's and 90's video game hardware, Curious Quail has evolved into more of a musician collective than a band proper with contributing members spanning the west coast of the United States from the deserts of the Coachella Valley to the Bay Area and the Pacific Northwest. Their mission has remained one of writing music that showcases an ongoing storyline told through lyrics and musical motifs while over engineering every technical decision from "how can we add more violin" to "yes the Gameboy needs an overdrive pedal". They're releasing two records this year; Empty Victories, and a nontraditional monthly writing exercise called Twelve Months, which is partially available on YouTube as you read this.
(Photo Credit: Loni Lonzo / @shutterghost_ )


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Featured Men
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Nahfis Nichols #1448858
PO Box 759
Bigstone Gap, Virginia 24219
Let me start off by saying that I'm incarcerated on a wrongful conviction, and have been blessed to have had one of my two charges overturned (still awaiting the outcome of the second). It's not easy being locked up, let alone having to do it by yourself. Therefore, I'm looking for a friend, but am open to the possibility of something more. what I'm looking for is also what I bring. So if you want a good friendship with someone who will listen to you, allow you to express yourself, and understand you, I'm here! I'm a laid-back person. I like to laugh, I'm far from immature. I'm into history, working out and trying to better myself, each and every day. If you're interested in getting to know more about me, you can write me snail mail, or email me by going to JPay.
Race: Black
Date of Birth: 08-28-1990
Height: 5'10"
Earliest Release Date: 2065
Maximum Release Date: 2065
Would you like letters from both sexes? Women
(This does not refer to sexual orientation)
Education: GED
Occupation before prison:
Activities in prison: Working out, Reading
Can you receive and send emails: Yes via www.JPay.com
Notice: To verify all or some of the information
given please click here


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Taken during our visit to the Bodhnath Temple in Kathmandu. These we're picked from our hotel owners garden. Hot stuff!
Archive | Nepal
Our Homestay Experience at Sanu Guesthouse, Kathmandu
By Stuart Edwards in Blog, Nepal
If you want to get away from the crowds in Thamel, but still be in the Kathmandu area then Sanu Guesthouse is only a 15 minute taxi or bus ride away. Run by the friendly Sarita whose smile alone is worth coming for.
Travel Photo Essay: A Lunar Eclipse in Bodhnath, Nepal
Eavesdropping on some other travellers, we heard it would be insanity not to miss the full moon celebrations in Bodhnath.
Chitwan National Park, Nepal – Travel Photo Essay
The Chitwan National Park is a vast diverse landscape nearly 950 sq km in size. No trip to Nepal would be complete without a visit to one of the outstanding national parks.
← Previous 1 2


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You walk down the street and you see all these perfect and stylish moms with their perfect manicure and perfect hair, sporting really expensive clothes and heels all the time.
Then there's you.
I often feel like I have to be like the others to be accepted. Then I remember that I am in no way the kind of person that wants to fit in.
But sometimes it hurts to be scrutinised from head to toe by everybody, to be considered the weirdo all the time.
It's always been like this, especially during my hardcore punk phase with a 25cm mohawk. Now that I will be mom soon I wonder if the situation will worsen because a mom with fucked up hair, tattoos and "satanic" t-shirts are not to be trusted.
How to deal with the feeling of looking like an unfit parent? I honestly don't know how it's going to affect me since my baby bump is still so small you could tell it's food baby.
That's one thing I know and is that I already can feel the judgments that come even from family members about my baby outfit choices. Yeah you know black stuff, skeletons, and monsters are not suitable for an adult person, let alone a sweet cutie pie.
I already had to deal with the "Are you going to dress that poor baby with all crazy clothes, aren't you?"
And I'm already tired and it's only been 3 and a half months of pregnancy. Somebody save me!
So yeah, sometimes I think: "Should I normalise for my child's sake?" And the answer is "No, I won't."
When the funny looks will begin to bother me I will just smile and raise my middle finger maybe #jk


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While the world grows increasingly materialistic in nature, it's rare to find people who actually appreciate the effort of a gift than the quality of the gift. A gift necessarily doesn't need to have to have a high monetary value. It is the warmth and the wishes that are associated with it make it worth of value. All around the year, there are numerous occasions on which it has become rather necessary to present a gift with a high monetary value to maintain the social status and dignity among the peers. But occasions such as New Years where celebrations are mandatory, with the amazing people existing in everyone's life that sees through the monetary value of a gift and appreciate the effort. To present them with gifts of that wonderful nature, presenting them with a bouquet of flowers is the best option. Send New Year Flowers to the amazing people while partnering with us in the effort.
The flower has the essence to show love when words fail
A Flower has that the capacity to lift up the spirit of everyone just by its mere presence. The sweet fragrance and the bright colors can soothe the soul of a heartbroken person as well. Even though they aren't quite valuable in terms of money but a person who knows how to appreciate efforts will know the value of the gift. While there are innumerable things one could gift another, a bouquet of flowers could also be an additional gift as well. To send New Year flowers to the person all one has to do is to contact our specialized team with the specifications if any. Flowers can also be arranged on vases, baskets or as bouquets. Customizing as per the client requests are also done here. Maintain the awesome and creativity while sending them flowers. Make your people understand the uniqueness and the sensuality present in the flowers while expressing feelings that words fail to make understand. It is the effort to make a day special matters the most even without the presence of the person physically by sending New Years Flowers.
Send flowers with unique features and creativeness
People have a different taste all together, so having very few options might be a problem. Often people are allergic to quite a lot of them as well. Keeping all the problems in mind, our team has an amazing collection of flowers for everyone. All of the flowers are natural and chemical free ensuring the prettiness in it's structure. Flowers of different shades starting with red, yellow, purple, pink and white are available as well. The freshness is ensured by sending New Year flowers from the closest outlet. Send these lovely bouquets to the amazing people on the beginning of the year and see the happiness filling within them. The beautiful nature of the person symbolizes with the beautiful flowers. To make sure everyone receives the flowers on New Year's make your booking now and send new year flowers.
Goa, situated in the coastal area of Konkan, happens to be one of the most well-known states in South India. Dear to both the Indian and the foreign tourists, Goa is the visiting place for thousands every year. Landmarks, cultural heritages, festivities, all these have made Goa a site not to miss at any cost.
On Sunday 13 May it is Mother's Day again. Are you still looking for a nice present fo...Read more
Chocolate is a favourite food item that is adored by the people of all ages, from the infants to the aged ones. So it can be chosen as a perfect gift for any person, whom you may want to convey your special love and affection. Hyderabad is a large city that is inhabited by numerous people and this population has further increased due to the industrial growth in this metropolitan city, which has...Read more
So it's February and that time of the year again, the season of love! However, if you are confused about how you can make your other half to feel special and gift him something that's beyond the cliché of cards, chocolates, and flowers? Well, do not worry! Here's a list of four Valentines gifts that you can give him to make him feel special.
Personalized Gifts:
Personalized Gifts always makes ...Read more
Cakes are essentially a sweet pastry, which can be modified into different forms by the means of customizations. It is almost impossible to find a person, who does not prefer to have cakes. With flavors in plenty, cakes are just available in all the possible ways a person can think of customizing by the means of decorating and adding different candies and stuff. Undoubtedly, this is a wonderful...Read more
With Gomilio you can create public and private downloads areas. Upload your files and make them available to users of your site!


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Salinas: Outworking yesterday.
It's become a slogan, a pledge and a belief among a cast of unknowns.
"That's our identity," Salinas High girls basketball coach Casey Neligh said. "We block out distractions."
A year after winning just six games, the Cowboys will go into Mission Division play Thursday with an 8-4 mark and a wave of confidence.
"It's about us," Neligh said. "When we can focus on what we do, then outside influences don't effect us. I don't look too far into the future."
A promising future with a pair of freshman standouts to build a program off of arrived perhaps even sooner than Neligh expected.
A stretch of five consecutive wins included decisions over then undefeated Notre Dame and North Salinas.
"Beating North Salinas is the standard," sophomore Makenzi Laporte said. "But I was not surprised. What we have this year is cohesiveness, confidence and chemistry."
Yet, the game that may have pointed Salinas in the right direction was a three-point loss to Notre Dame of San Jose.
"To me, that was the turning point in terms of confidence," senior Christina Chagnon said. "We lost, but we knew we had something. We worked so well. The team energy has been great."
One of two senior holdovers on this revamped roster, Chagnon has taken on a leadership role along with Neveah Pinon, embracing the youth on the team, showcasing work ethics that have become contagious.
"Team chemistry is the biggest difference," Chagnon said. "There was more separation last year. We were more accepting to the new freshmen. We all have the same goals."
Measuring improvement last season came in simple steps for the Cowboys. Neligh wanted to install an offense in his first season that he believed could develop players over the course of four years.
Instead of wins, Neligh looked for improvement in practices, or quarters during a game. Trusting the process meant testing patience and not going away from his plan.
"If I'm confident, my team is confident," Neligh said. "We've raised the basketball IQ. I stuck to my guns with this read and react offense. We're playing basketball the way the game should be."
It's hard to argue with the results in the team's start. Two of those losses were by five points or less. Two have come against Seaside.
When gauging improvement, the battles with Seaside are a clear example of a program on the right path.
Salinas fell by 29 points to the reigning Gabilan Division champion Spartans on Dec. 1. Fourteen days later it lost by just five points.
"You can always control how you play defensively," Neligh said. "Having film of Seaside, I felt we came up with a good defensive scheme. We limited transition 3s. And we've gotten better as a team."
Neligh, a member of The Herald's All-County football and basketball teams in 2001 at Salinas, has coaching pedigree in his background.
His grandfather is Cork Haddan, arguably one of the most successful basketball coaches in the Salinas Valley, and someone Neligh has watched coach basketball since he was in the second grade.
"I watched how he spoke, commanded his team," Neligh said. "When you are around this your entire life, things rub off. I've surrounded myself with a plethora of basketball knowledge with my assistants. I love what I do."
Neligh, a special education instructor at Salinas, believed he could develop his program around the likes of Chagnon, Pinon and Laporte.
What Neligh didn't know until the summer was freshmen Jenika Zurita and Brooke Cannon were going to be a part of the change in direction for the program.
"I didn't know who they were until the summer," Neligh said. "They played with our summer league team. When I got my hands on them, it was a no brainer to bring them up."
Zurita has become the floor leader for Salinas, igniting the offense with tenacious defense and the ability to get the ball to her teammates.
"She is the floor general," Neligh said. "She orchestrates the offense and gets the ball to our shooters. Her points come from the defensive side of the ball on transition buckets."
Cannon has provided Salinas with a threat on the perimeter, complimenting Laporte's talents in the paint.
"Brooke is probably one of the purest shooters on the team," Neligh said. "She can really shoot the ball. And she's just a workhorse."
So is the 5-foot-10 Laporte, who plants herself in the paint. She's become the heart of the team's offense, averaging a double-double per game on the season.
"She did not like the post," Neligh said. "Probably still doesn't. But she's embraced her role. She can score from any spot on the floor. She does whatever the team needs."
Laporte didn't dispute Neligh's notion about her playing in the post.
"He's right, I don't like it," Laporte said. "I see myself on the outside. But it has grown on me. We're not a big team. For me, it's about what's best for the team."
That includes the defensive side of the ball where Neligh pointed toward Laporte's fearlessness in taking a charge.
"When you can get a scorer to take a charge, there is no bigger momentum change," Neligh said. "Her game is still evolving."
What Salinas has this season is depth. Kayla Stone and Leslie Cital have been contributors, while Isabella Perez came out as a senior after three years of soccer to provide what Neigh calls the X-factor.
"Rebounding and enforcement," said Neligh, when speaking of Perez, who led the soccer team in saves in goal the past two years.
As young as the Cowboys are, Neligh points back to Chagnon and Nevah in providing the leadership on the hardwood.
"They are the landlords of this basketball team," Neligh said. "They collect the rent. There is a true tradition with these seniors. The teams respect our captains."
That, in turn, has given Salinas something it was lacking last year in a connection on and off the court.
"We began to build that bond during the summer league at MPC," said Chagnon, a member of The Herald's All-County volleyball team last fall. "We brought the freshmen in with open arms."
The next step is playing at a high level for 32 minutes. The youth and inexperience still creep up from time to time, as evident in a 39-30 loss to Notre Dame on Tuesday.
"We're still looking for consistency," Neligh said. "We have lapses. It's part of our growth. You have to maintain goals and priorities."


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Ian Hobson
(Photo: Maggie Tang)
Ian lives in Berlin, Germany and is the Technical Principal for DSP at Ableton, where he also works as a software developer on the instruments and effects included in Ableton Live. He moved from the UK in 2011 after receiving an MSc in Digital Music Processing from the Centre for Digital Music at Queen Mary, University of London. He also holds a BA in Music Technology from De Montfort University, Leicester.
How do you describe yourself (e.g. are you a performer, a composer, a technologist, an engineer, some combination of these or, indeed, something else) and why?
I'm a software developer who builds tools for musicians. When people ask, I say "I help people make weird noises", but what I really hope that I'm doing is helping people to express themselves more freely. Having a musical idea and then finding a way to hear it coming out of your speakers is a wonderful feeling, and I enjoy being part of a movement towards making that easier to achieve. I'm not one of those people who believes that making music has to involve in-the-moment effort or pain; I don't need to see a musician suffer for me to be able to enjoy their music. The more expressive a musician can be, the better they're able to communicate their emotions to the listener, and more fluid expression generally results in more satisfying music. If a musician wants their compositions or performances to involve great effort, then I'd prefer that their labour goes into creation, exploration, expression, and not fighting against their tools. I see a large part of my work as helping to increase the expressivity of music software to make creativity easier for musicians.
Why do you make music?
Before I became a developer, I tended to make music that tried to communicate a very specific idea. For me, making a piece of music was like a puzzle that had to be solved, with all the pieces needing to be slotted into place logically and methodically. There's nothing inherently wrong with this approach to composition, but for me it was driven from a desire to solve problems, and over time I found that my problem-solving impulses were satisfied by my work in building software, which then altered my relationship to composition. Nowadays, I tend to make music as a hobby, to relax, and to maybe work through some thoughts and emotions. I'm much more comfortable with enjoying a musical moment for its own sake than I was in the past.
What music do you make?
I play a lot of piano, recording improvisations and then pull out ideas which I usually rework with synthesizers, and layer themes together to try and capture the mood I'm in. This approach allows me to separate the initial creative moments from the editing and arrangement parts of the process, which tend to pull me into a more analytical frame of mind. Recently I've been playing a lot with feedback loops, enjoying the tones and textures that you can uncover when running audio in a loop through a ton of effects. It's loads of fun and the sonic potential is limitless, so it's great for open-ended exploration.
Do you have any other useful or relevant things to say about being a musician in the digital age?
There are so many powerful tools readily available now to us as musicians that it's easy to become fatigued when trying to decide where to begin, what sounds and effects to use, how far to go when arranging, how to decide when you've finished... The possibilities are endless, so it's very important to not allow technology to overwhelm you, but instead to harness it to inspire and empower you. As someone who works on music software, I'm aiming to continue to make tools easier to use and as expressive as possible. We're trying to solve a lot of problems with the current state of music technology, and it might be frustrating for a musician to know that the technology available to them will be better tomorrow. Perhaps it helps to keep in mind that technology is the best that it's ever been *today*, so it's good to embrace what we have now, and make some music with it. The same acceptance of availability can be applied to your own knowledge of technology. If you're feeling limited, then learn some new tools, but if you're feeling creative then stick to what you know well, stay in the moment and make the most out of it.


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CAA News Today
News from the Art and Academic Worlds
posted by CAA — Oct 10, 2018
JPMorgan Chase Gives $300,000 to Theaster Gates's Rebuild Foundation in Chicago
The foundation will put the funds toward the final phase of renovation for its Arts and Innovation Incubator. (ARTnews)
'Conventional Narratives of History Are Being Expanded': Native Art Is Now Appearing in the Met's American Wing
A new exhibition at the Metropolitan Museum of Art marks the first time the museum has held a show of Native American art in its American wing. (artnet News)
Tania Bruguera on Transforming Tate Modern's Turbine Hall
"We live in a time when we need to defend complexity and the right to be complex." (Apollo Magazine)
Tackling Harassment and its Roots in Scholarly Communication: Practical Steps for Organizations and Individuals
How can we move to the practical level of making real change as individuals? (Scholarly Kitchen)
The Morality Wars: Should Art be a Battleground for Social Justice?
"The defining objective of the civil rights movement and the women's movement and the gay rights movement was equality, sure, but also motion—forward, upward, outward. The country has never looked both more and less like what these movements aspired to achieve." (New York Times Magazine)
Diversity Fatigue Is Real
It is the very people who are the most committed to doing diversity work who are experiencing "diversity fatigue." (Chronicle of Higher Education)


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Got it! The piyaz organized from the Antalya province has tahini (sesame seed paste) mixed to the sauce to help make an emulsion. Just how much or how very little tahini you utilize is down to private flavor. The Aksu piyaz was, for us, spot on!
The 2 rounded towers you'll be able to see previously mentioned are huge and therefore are the one survivors from the Hellenistic time period. (The theatre was also built-in the Hellenistic period but has become a mixture of Hellenistic and Roman development). Walk from the gates, outside of a horseshoe-shaped courtyard and ultimately, you bump into the Roman baths.
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By tram: The new AntRay is the key gentle-rail line, a streamlined support linking the primary bus station with other parts of the city. In contrast, the Considerably smaller sized Nostaljic Tramvay passes various historic websites and the most crucial beaches, together the resort's boulevard.
The final suggestion of things to accomplish in Antalya, really starts in that region but finishes in Fethiye, even more alongside, the coastline. The Lycian Way is among the planet's greatest long walks and is particularly 320 miles prolonged.
After that initially Antalya trip as well as the unforgettable prevent off in Korkuteli, I followed many Turkish white bean salad recipes. They had been fantastic. They were being the traditional Turkish piyaz servings we've considering that relished on our travels in places including Köfteci Ahmet in Eskişehir and Edirnekapı in Istanbul.
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Turkey has taken Yet another move in eco-friendly tourism. The state has zoned large regions of coastline as no-build regions. I know a tranquil bay where a resort built devoid of permission was demolished. In February, Turkey signed the Kyoto settlement to cut CO2 emissions. Now the Calista Vacation resort within the pine forests at Belek, outside the house Antalya, has won a Environmentally friendly Star award for ecofriendly accommodation.
Top Guidelines Of antalya things to do
No holiday getaway to Fethiye, Turkey is full and not using a pay a visit to to Ölüdeniz; one of the most photographed shorelines on the planet. Belcekiz Seaside has the crashing waves or you can swim from the lagoon. The dolmuş from Fethiye will get you all the way down to the non-public seashores of the lagoon at no cost for those who request the driving force.


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Ecru Cami and Shorties Sleepwear Set
This cute ecru sleepwear set from the Pearl'amore collection by Lume di Luna comprises a cami top and matching shorts with an eyelash lace trim.
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Product Description
Material: Polyester/Rayon.
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Additional Information
Additional Information
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Novato Healthcare Center
1565 Hill Road, Novato, CA 94947
Novato Healthcare Center is part of the for-profit Brius chain dozens of nursing homes throughout California. Novato Healthcare Center workers voted to join NUHW in 2015.
Alex Early
(617) 816-4260
Novato Healthcare Center RECENT LEAFLETS
Novato Healthcare Center LATEST NEWS
NUHW Pulse – April 2017: the latest news from NUHW
POSTED ON April 15th, 2017
Welcome to the April 2017 edition of NUHW Pulse, our union's e-newsletter! Dozens of Keck USC workers join NUHW More than 60 workers at Keck Medical Center at the University of Southern California voted overwhelmingly to join NUHW in the past month. NUHW now represents more than 1,000 healthcare workers at USC. Read more Members can count […]
Dozens of workers at Keck USC join NUHW
POSTED ON April 12th, 2017
More than 60 workers at Keck Medical Center at the University of Southern California voted overwhelmingly to join NUHW in the past month. NUHW now represents more than 1,000 workers at two USC hospitals — Keck Medical Center and Norris Cancer Hospital. On April 12, 17 workers at Keck's Beverly Hills clinic voted 94 percent to […]
Taking part in the resistance
POSTED ON April 12th, 2017
Throughout the state, scores of NUHW members are engaged in the resistance movement to defend working people, immigrants, and an equitable healthcare system. NUHW members' commitment was on full display at many town halls in recent months. At Congressmember Barbara Lee's joint town hall with Alameda County Supervisor Wilma Chan, dozens of workers from UCSF […]
Brius Healthcare under fire again
POSTED ON April 10th, 2017
California's largest nursing home company, Brius Healthcare, continues to come under fire for its abysmal patient care record and unscrupulous profit-driven approach to healthcare. As reported in the Eureka Times-Standard, in late March the families of two men who died after developing pressure sores in Brius nursing homes filed wrongful death lawsuits against the […]
April 2017 – Around the union
POSTED ON April 10th, 2017
Read more below for the latest developments in the union, including grievance victories, settlements, and bargaining updates. Sterile processing techs at Napa's Queen of the Valley organized their co-workers in other departments to join them in a march on the boss to address chronic understaffing and poor communications at the hospital. In their meeting with […]


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Alexander Anufriev has a personal and sincere gaze at cold Russia
This series is both a subjective look at aspects of contemporary life in Russia and a visual dictionary of a whole country. For me it's about finding out what defines contemporary Russia, that's why besides subjective interests and national cliches I do visualise significant social processes that takes place during last years
Caleb Churchill and the contradictions of our contemporary environment
Our environment, as it appears to us in photographs, is translated as landscape, a landscape that is transformed, altered and romanticized by American culture. Despite a desire for the unspoiled ideal of the American landscape that prompted the founding of the National Park system, our human need to organize, facilitate and take ownership, has become the geographical reality.
Jessica Wolfelsperger captures the mystic journey back to her homeland
In search of the experience of a divine or absolute reality, I discover and explore my homeland new and wash myself free from my daily life in the bustling metropolis and devise my own legend – according to the life philosophy of Monte Verità.
The cradle of flavour and quiet: Bottega Culinaria by Cinzia Mancini
Discovering Abruzzo through its people.


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