Sarah Song

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Abstract There remain no clear guidelines for the optimal management of patients with metastatic breast cancer. To better understand its natural history, we undertook a detailed examination of 197 autopsies performed on women who died of breast cancer. We reviewed clinical, treatment and pathological aspects of all cases and, additionally, pathological features and biomarker expression ( ER , PgR , HER2 , EGFR , p53, Ki67, c‐Kit, CK AE1 / AE3 ) were assessed in detail for the primary tumour and matched metastases for 55 of the cases. Genomes of the primary tumour and multiple metastases were analysed by array‐based comparative genomic hybridization for six cases ## . 945 metastatic deposits were identified, with a median of four/patient. The most common organs involved were lung/pleura (80%), bone (74%), liver (71%) and non‐axillary lymph nodes (55%). Major findings included: (a) patients with CNS metastases were more likely to have bone metastases ( p < 0.013); (b) younger age was associated with metastasis to the liver (≤ 49 years; p < 0.001) and to gynaecological organs (≤ 49 years; p = 0.001); (c) surgical excision of the primary tumour was associated with metastasis to the liver ( p = 0.002); and (d) ER and PgR showed down‐regulation during progression in a non‐random manner, particularly in lung/pleura ( ER ; p < 0.001), liver and bone metastases. Genomic analysis revealed DNA copy number variation between the primary tumour and metastases ( e.g. amplification of 2q11.2–q12.1 and 10q22.2–q22.3) but little variation between metastases from the same patient. In summary, the association of CNS and bone metastases, liver and gynaecological metastases in young women and the risk of liver metastases following surgery have important implications for the management of patients with breast cancer. Clonal heterogeneity of the primary tumour is important in developing metastatic propensity and the change in tumour phenotype during progression/colonization highlights the importance of sampling metastatic disease for optimal treatment strategies. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Calcium signaling is a critical regulator of cell proliferation. Elevated expression of calcium channels and pumps is a characteristic of some cancers, including breast cancer. We show that the plasma membrane calcium channel TRPV6, which is highly selective for Ca(2+), is overexpressed in some breast cancer cell lines. Silencing of TRPV6 expression in a breast cancer cell line with increased endogenous TRPV6 expression leads to a reduction in basal calcium influx and cellular proliferation associated with a reduction in DNA synthesis. TRPV6 gene amplification was identified as one mechanism of TRPV6 overexpression in a subset of breast cancer cell lines and breast tumor samples. Analysis of two independent microarray expression datasets from breast tumor samples showed that increased TRPV6 expression is a feature of estrogen receptor (ER)-negative breast tumors encompassing the basal-like molecular subtype, as well as HER2-positive tumors. Breast cancer patients with high TRPV6 levels had decreased survival compared with patients with low or intermediate TRPV6 expression. Our findings suggest that inhibitors of TRPV6 may offer a novel therapeutic strategy for the treatment of ER-negative breast cancers.

Insomnia is a common and debilitating disorder experienced by cancer survivors. Although cancer survivors express a preference for using nonpharmacological treatment to manage insomnia, the comparative effectiveness between acupuncture and Cognitive Behavioral Therapy for Insomnia (CBT-I) for this disorder is unknown.This randomized trial compared 8 weeks of acupuncture (n = 80) and CBT-I (n = 80) in cancer survivors. Acupuncture involved stimulating specific points on the body with needles. CBT-I included sleep restriction, stimulus control, cognitive restructuring, relaxation training, and education. We measured insomnia severity (primary outcome), pain, fatigue, mood, and quality of life posttreatment (8 weeks) with follow-up until 20 weeks. We used linear mixed-effects models for analyses. All statistical tests were two-sided.The mean age was 61.5 years and 56.9% were women. CBT-I was more effective than acupuncture posttreatment (P < .001); however, both acupuncture and CBT-I produced clinically meaningful reductions in insomnia severity (acupuncture: -8.31 points, 95% confidence interval = -9.36 to -7.26; CBT-I: -10.91 points, 95% confidence interval = -11.97 to -9.85) and maintained improvements up to 20 weeks. Acupuncture was more effective for pain at the end of treatment; both groups had similar improvements in fatigue, mood, and quality of life and reduced prescription hypnotic medication use. CBT-I was more effective for those who were male (P < .001), white (P = .003), highly educated (P < .001), and had no pain at baseline (P < .001).Although both treatments produced meaningful and durable improvements, CBT-I was more effective and should be the first line of therapy. The relative differences in the comparative effectiveness between the two interventions for specific groups should be confirmed in future adequately powered trials to guide more tailored interventions for insomnia.

Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value=0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value=0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

Triple-negative breast cancer (TNBC) is associated with poor survival. Chemotherapy is the only standard treatment for TNBC. The prevalence of BRCA1 inactivation in TNBC has rationalized clinical trials of poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors. Similarly, the overexpression of epidermal growth factor receptor (EGFR) rationalized anti-EGFR therapies in this disease. However, clinical trials using these 2 strategies have not reached their promise. In this study, we used EGFR as a target for radioimmunotherapy and hypothesized that EGFR-directed radioimmunotherapy can deliver a continuous lethal radiation dose to residual tumors that are radiosensitized by PARP inhibitors and chemotherapy.We analyzed EGFR messenger RNA in published gene expression array studies and investigated EGFR protein expression by immunohistochemistry in a cohort of breast cancer patients to confirm EGFR as a target in TNBC. Preclinically, using orthotopic and metastatic xenograft models of EGFR-positive TNBC, we investigated the effect of the novel combination of (177)Lu-labeled anti-EGFR monoclonal antibody, chemotherapy, and PARP inhibitors on cell death and the survival of breast cancer stem cells.In this first preclinical study of anti-EGFR radioimmunotherapy in breast cancer, we found that anti-EGFR radioimmunotherapy is safe and that TNBC orthotopic tumors and established metastases were eradicated in mice treated with anti-EGFR radioimmunotherapy combined with chemotherapy and PARP inhibitors. We showed that the superior response to this triple-agent combination therapy was associated with apoptosis and eradication of putative breast cancer stem cells.Our data support further preclinical investigations toward the development of combination therapies using systemic anti-EGFR radioimmunotherapy for the treatment of recurrent and metastatic TNBC.

Paget's disease is a focal condition of bone. To study changes in cells within pagetic lesions, we cultured osteoblasts and stromal cells from 22 patients and compared gene expression in these cells to cells from healthy bone. We identified several differentially regulated genes, and we suggest that these changes could lead to the formation of the lesions.Paget's disease is a focal condition of bone of unknown cause. Although it is regarded as primarily an osteoclast disorder, the tight coupling of the activity of osteoclasts and osteoblasts suggests that the osteoblast could play a key role in its pathogenesis. The aim of the study was to identify possible changes in pagetic osteoblasts and stromal cells that might contribute to the development of pagetic lesions.Candidate genes were identified based on known bone cell regulators, supplemented with microarray analysis. Gene expression was determined by real-time PCR in primary cultures of osteoblasts and bone marrow stromal cells from pagetic patients and control subjects. Concentrations of secreted proteins were determined by ELISA.Dickkopf1 mRNA and protein levels were increased in both pagetic osteoblast and stromal cell cultures, and interleukin (IL)-1 and IL-6 were overexpressed in pagetic osteoblasts. These changes parallel recent findings in myeloma bone disease, which shares some clinical similarities with Paget's disease. Alkaline phosphatase was overexpressed, and bone sialoprotein and osteocalcin were underexpressed in pagetic osteoblasts, consistent with their circulating levels in pagetic patients. It is hypothesized that overexpression of Dickkopf1, IL-1, and IL-6 would result in stimulation of osteoclast proliferation and inhibition of osteoblast growth, leading to the development of the characteristic lytic bone lesions. By stimulating osteoblast differentiation, Dickkopf1 and IL-6 may also promote mineralization, leading to the conversion of lytic lesions to sclerotic.These findings suggest that dysregulated gene expression in pagetic osteoblasts could cause the changes in bone cell number and function characteristic of Paget's disease.

The analysis of gene sets has become a popular topic in recent times, with researchers attempting to improve the interpretability and reproducibility of their microarray analyses through the inclusion of supplementary biological information. While a number of options for gene set analysis exist, no consensus has yet been reached regarding which methodology performs best, and under what conditions. The goal of this work was to examine the performance characteristics of a collection of existing gene set analysis methods, on both simulated and real microarray data sets. Of particular interest was the potential utility gained through the incorporation of inter-gene correlation into the analysis process. Each of six gene set analysis methods was applied to both simulated and publicly available microarray data sets. Overall, the various methodologies were all found to be better at detecting gene sets that moved from non-active (i.e., genes not expressed) to active states (or vice versa), rather than those that simply changed their level of activity. Methods which incorporate correlation structures were found to provide increased ability to detect altered gene sets in some settings. Based on the results obtained through the analysis of simulated data, it is clear that the performance of gene set analysis methods is strongly influenced by the features of the data set in question, and that methods which incorporate correlation structures into the analysis process tend to achieve better performance, relative to methods which rely on univariate test statistics.

Our understanding of breast cancer heterogeneity at the protein level is limited despite proteins being the ultimate effectors of cellular functions.We investigated the heterogeneity of breast cancer (41 primary tumors and 15 breast cancer cell lines) at the protein and phosphoprotein levels to identify activated oncogenic pathways and developing targeted therapeutic strategies.Heterogeneity was observed not only across histological subtypes, but also within subtypes.Tumors of the Triple negative breast cancer (TNBC) subtype distributed across four different clusters where one cluster (cluster ii) showed high deregulation of many proteins and phosphoproteins.The majority of TNBC cell lines, particularly mesenchymal lines, resembled the cluster ii TNBC tumors.Indeed, TNBC cell lines were more sensitive than non-TNBC cell lines when treated with targeted inhibitors selected based on upregulated pathways in cluster ii.In line with the enrichment of the upregulated pathways with oncoclients of Hsp90, we found synergy in combining Hsp90 inhibitors with several kinase inhibitors, particularly Erk5 inhibitors.The combination of Erk5 and Hsp90 inhibitors was effective in vitro and in vivo against TNBC leading to upregulation of pro-apoptotic effectors.Our studies contribute to proteomic profiling and improve our understanding of TNBC heterogeneity to provide therapeutic opportunities for this disease.

Background Artemisinin resistance observed in Southeast Asia threatens the continued use of artemisinin-based combination therapy in endemic countries. Additionally, the diversity of chemical mode of action in the global portfolio of marketed antimalarials is extremely limited. Addressing the urgent need for the development of new antimalarials, a chemical class of potent antimalarial compounds with a novel mode of action was recently identified. Herein, the preclinical characterization of one of these compounds, ACT-451840, conducted in partnership with academic and industrial groups is presented. Method and Findings The properties of ACT-451840 are described, including its spectrum of activities against multiple life cycle stages of the human malaria parasite Plasmodium falciparum (asexual and sexual) and Plasmodium vivax (asexual) as well as oral in vivo efficacies in two murine malaria models that permit infection with the human and the rodent parasites P. falciparum and Plasmodium berghei, respectively. In vitro, ACT-451840 showed a 50% inhibition concentration of 0.4 nM (standard deviation [SD]: ± 0.0 nM) against the drug-sensitive P. falciparum NF54 strain. The 90% effective doses in the in vivo efficacy models were 3.7 mg/kg against P. falciparum (95% confidence interval: 3.3–4.9 mg/kg) and 13 mg/kg against P. berghei (95% confidence interval: 11–16 mg/kg). ACT-451840 potently prevented male gamete formation from the gametocyte stage with a 50% inhibition concentration of 5.89 nM (SD: ± 1.80 nM) and dose-dependently blocked oocyst development in the mosquito with a 50% inhibitory concentration of 30 nM (range: 23–39). The compound’s preclinical safety profile is presented and is in line with the published results of the first-in-man study in healthy male participants, in whom ACT-451840 was well tolerated. Pharmacokinetic/pharmacodynamic (PK/PD) modeling was applied using efficacy in the murine models (defined either as antimalarial activity or as survival) in relation to area under the concentration versus time curve (AUC), maximum observed plasma concentration (Cmax), and time above a threshold concentration. The determination of the dose–efficacy relationship of ACT-451840 under curative conditions in rodent malaria models allowed prediction of the human efficacious exposure. Conclusion The dual activity of ACT-451840 against asexual and sexual stages of P. falciparum and the activity on P. vivax have the potential to meet the specific profile of a target compound that could replace the fast-acting artemisinin component and harbor additional gametocytocidal activity and, thereby, transmission-blocking properties. The fast parasite reduction ratio (PRR) and gametocytocidal effect of ACT-451840 were recently also confirmed in a clinical proof-of-concept (POC) study.

Chronic inflammation has been a proposed mechanism of resistance to aromatase inhibitors in breast cancer. Stratifying by HER2 status, a matched case-control study from the Wellness After Breast Cancer-II cohort was performed to assess whether or not elevated serum inflammatory biomarkers (C-Reactive protein [CRP], interleukin-6 [IL-6], and serum amyloid A [SAA]) and/or the presence of a high-risk IL-6 promoter genotype were associated with recurrence of hormone receptor positive (HR+) early breast cancer. Estrogen levels were also measured and correlated with biomarkers and disease outcomes. CRP and SAA were significantly associated with an increased risk of recurrence in the HR+/HER2- group, but not the HR+/HER2+ group. Mean serum estrogen levels were non-significantly elevated in patients who relapsed vs. non-relapsed patients. Surprisingly, high-risk IL-6 promoter polymorphisms were strongly associated with HER2+ breast cancer relapse, which has potential therapeutic implications, as elevated intracellular IL-6 has been associated with trastuzumab resistance in pre-clinical models.

Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression alterations can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing high-level gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immunohistochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where fresh frozen material is scarce.

Patient preference is an essential component of patient-centered supportive cancer care; however, little is known about the factors that shape preference for treatment. This study sought to understand what factors may contribute to patient preference for two non-pharmacological interventions, acupuncture or cognitive behavioral therapy for insomnia (CBT-I). We conducted individual, open-ended, semi-structured interviews among cancer survivors who had completed active treatment and met the diagnostic criteria for insomnia disorder. Two forms of codes were used for analysis: a priori set of codes derived from the key ideas and a set of codes that emerged from the data. Among 53 participants, the median age was 60.7 (range 27–83), 30 participants (56.6%) were female, and 18 (34%) were non-white. We identified three themes that contributed to an individual’s treatment preference: perception of the treatment’s evidence base, experience with the treatment, and consideration of personal factors. Participants gave preference to the treatment perceived as having stronger evidence. Participants also reflected on positive or negative experiences with both of the interventions, counting their own experiences, as well as those of trusted sources. Lastly, participants considered their own unique circumstances and factors such as the amount of work involved, fit with personality, or fit with their “type” of insomnia. Knowledge of the evidence base, past experience, and personal factors shaped patient preference regardless of whether they accurately represent the evidence. Acknowledging these salient factors may help inform patient-centered decision-making and care.

Long COVID (LongC) is associated with a myriad of symptoms including cognitive impairment. We reported at the beginning of the COVID-19 pandemic that neuronal-enriched or L1CAM+ extracellular vesicles (nEVs) from people with LongC contained proteins associated with Alzheimer’s disease (AD). Since that time, a subset of people with prior COVID infection continue to report neurological problems more than three months after infection. Blood markers to better characterize LongC are elusive. To further identify neuronal proteins associated with LongC, we maximized the number of nEVs isolated from plasma by developing a hybrid EV Microfluidic Affinity Purification (EV-MAP) technique. We isolated nEVs from people with LongC and neurological complaints, AD, and HIV infection with mild cognitive impairment. Using the OLINK platform that assesses 384 neurological proteins, we identified 11 significant proteins increased in LongC and 2 decreased (BST1, GGT1). Fourteen proteins were increased in AD and forty proteins associated with HIV cognitive impairment were elevated with one decreased (IVD). One common protein (BST1) was decreased in LongC and increased in HIV. Six proteins (MIF, ENO1, MESD, NUDT5, TNFSF14 and FYB1) were expressed in both LongC and AD and no proteins were common to HIV and AD. This study begins to identify differences and similarities in the neuronal response to LongC versus AD and HIV infection.

High temperature requirement A1 (HTRA1) is a member of the serine protease family, comprising four structural domains: IGFBP domain, Kazal domain, protease domain and PDZ domain. HTRA1 encodes a serine protease, a secreted protein that is widely expressed in the vasculature. HTRA1 regulates a wide range of physiological processes through its proteolytic activity, and is also involved in a variety of vascular abnormalities-related diseases. This article reviews the role of HTRA1 in the development of vascular abnormalities-related hereditary cerebral small vessel disease (CSVD), age-related macular degeneration (AMD), tumors and other diseases. Through relevant research advances to understand the role of HTRA1 in regulating signaling pathways or refolding, translocation, degradation of extracellular matrix (ECM) proteins, thus directly or indirectly regulating angiogenesis, vascular remodeling, and playing an important role in vascular homeostasis, further understanding the mechanism of HTRA1's role in vascular abnormality-related diseases is important for HTRA1 to be used as a therapeutic target in related diseases.

Heart disease is the major cause of death in diabetes, a disorder characterized by chronic hyperglycemia and cardiovascular complications. Diabetic cardiomyopathy (DCM) is increasingly recognized as a major contributor to diastolic dysfunction and heart failure in diabetes, but its molecular basis has remained obscure, in part because of its multifactorial origins. Here we employed comparative transcriptomic methods with quantitative verification of selected transcripts by reverse transcriptase quantitative PCR to characterize the molecular basis of DCM in rats with streptozotocin-induced diabetes of 16-wk duration. Diabetes caused left ventricular disease that was accompanied by significant changes in the expression of 1,614 genes, 749 of which had functions assignable by Gene Ontology classification. Genes corresponding to proteins expressed in mitochondria accounted for a disproportionate number of those whose expression was significantly modified in DCM, consistent with the idea that the mitochondrion is a key target of the pathogenic processes that cause myocardial disease in diabetes. Diabetes also induced global perturbations in the expression of genes regulating cardiac fatty acid metabolism, whose dysfunction is likely to play a key role in the promotion of oxidative stress, thereby contributing to the pathogenesis of diabetic myocardial disease. In particular, these data point to impaired regulation of mitochondrial β-oxidation as central in the mechanisms that generate DCM pathogenesis. This study provides a comprehensive molecular snapshot of the processes leading to myocardial disease in diabetes.

Aberrant promoter DNA methylation has been reported in childhood acute lymphoblastic leukaemia (ALL) and has the potential to contribute to its onset and outcome. However, few reports demonstrate consistent, prevalent and dense promoter methylation, associated with tumour-specific gene silencing. By screening candidate genes, we have detected frequent and dense methylation of the TESTIN (TES) promoter.Bisulfite sequencing showed that 100% of the ALL samples (n = 20) were methylated at the TES promoter, whereas the matched remission (n = 5), normal bone marrow (n = 6) and normal PBL (n = 5) samples were unmethylated. Expression of TES in hyperdiploid, TEL-AML+, BCR-ABL+, and E2A-PBX+ subtypes of B lineage ALL was markedly reduced compared to that in normal bone marrow progenitor cells and in B cells. In addition TES methylation and silencing was demonstrated in nine out of ten independent B ALL propagated as xenografts in NOD/SCID mice.In total, 93% of B ALL samples (93 of 100) demonstrated methylation with silencing or reduced expression of the TES gene. Thus, TES is the most frequently methylated and silenced gene yet reported in ALL. TES, a LIM domain-containing tumour suppressor gene and component of the focal adhesion complex, is involved in adhesion, motility, cell-to-cell interactions and cell signalling. Our data implicate TES methylation in ALL and provide additional evidence for the involvement of LIM domain proteins in leukaemogenesis.

Differential expression analysis of (individual) genes is often used to study their roles in diseases. However, diseases such as cancer are a result of the combined effect of multiple genes. Gene products such as proteins seldom act in isolation, but instead constitute stable multi-protein complexes performing dedicated functions. Therefore, complexes aggregate the effect of individual genes (proteins) and can be used to gain a better understanding of cancer mechanisms. Here, we observe that complexes show considerable changes in their expression, in turn directed by the concerted action of transcription factors (TFs), across cancer conditions. We seek to gain novel insights into cancer mechanisms through a systematic analysis of complexes and their transcriptional regulation. We integrated large-scale protein-interaction (PPI) and gene-expression datasets to identify complexes that exhibit significant changes in their expression across different conditions in cancer. We devised a log-linear model to relate these changes to the differential regulation of complexes by TFs. The application of our model on two case studies involving pancreatic and familial breast tumour conditions revealed: (i) complexes in core cellular processes, especially those responsible for maintaining genome stability and cell proliferation (e.g. DNA damage repair and cell cycle) show considerable changes in expression; (ii) these changes include decrease and countering increase for different sets of complexes indicative of compensatory mechanisms coming into play in tumours; and (iii) TFs work in cooperative and counteractive ways to regulate these mechanisms. Such aberrant complexes and their regulating TFs play vital roles in the initiation and progression of cancer. Complexes in core cellular processes display considerable decreases and countering increases in expression, strongly reflective of compensatory mechanisms in cancer. These changes are directed by the concerted action of cooperative and counteractive TFs. Our study highlights the roles of these complexes and TFs and presents several case studies of compensatory processes, thus providing novel insights into cancer mechanisms.

The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44 K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE–FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

The use of formalin fixed paraffin embedded (FFPE) tissues for DNA analysis has been met with great difficulties due to the degradative effect this fixation, processing and storage has on DNA quality. The lack of a suitable protocol to enhance the quality of such degraded DNA has been a great hindrance to our ability to make full use of clinically annotated FFPE cancer tissue banks. In this report we have begun to investigate the effectiveness and limitations to Illumina’s recent platform for the restoration of DNA derived from archival FFPE tissues. With the exception of select fresh frozen and blood samples, all DNA samples were extracted from FFPE tissues and restored according to Illumina’s protocol. The quality of the FFPE extracted DNA was then assessed by Illumina’s PCR-based quality control assay (QC-PCR) and the resultant DNA was subsequently run on Illumina’s SNP-based CGH chips. Chip call rates were largely used in order to determine the quality of a particular array. Overall, the chip data were highly reproducible as determined by comparing several technical replicate samples. FFPE-extracted and restored DNA performed well in comparison to DNA extracted from fresh frozen tumor and blood from the same patients, meeting the minimum standard for continuation of this platform. It should also be noted that the quality of the chip data was not appreciably enhanced by the use of sodium thiocyanate during the extraction of DNA from FFPE tissues. Moreover, chip quality was significantly lower, with regards to call rates and ‘quality’ of b allele frequency plots, when the recommended Roche DNA extraction kit was used instead of the Qiagen DNA extraction kit. Of great importance, we found that the QC-PCR provided an accurate prediction of chip quality as determined by comparing chip call rates with PCR signals derived from proprietary primer sets (rho= 0.6242, p<0.0001). The selection of restored DNA for future studies will be guided by the results from the QC-PCR assay. These preliminary data demonstrate the promise of Illumina’s DNA restoration protocol for FFPE extracted DNA from tissues older than three decades. Further studies are required to determine the full potential of this method for SNP-based CGH analysis of large FFPE tumor banks.

10001 Background: Insomnia is a common and debilitating disorder experienced by up to 60% of cancer survivors. We evaluated the effectiveness of acupuncture versus cognitive behavior therapy for insomnia (CBT-I). Methods: We conducted a randomized clinical trial of acupuncture vs. CBT-I among 160 post-treatment cancer survivors with clinically diagnosed insomnia disorder. Acupuncture involved stimulating body points with needles. CBT-I included sleep restriction, stimulus control, cognitive restructuring, relaxation training, and education. The intervention duration was eight weeks (primary end point) with a follow up assessment at 20 weeks. Insomnia severity, measured by the Insomnia Severity Index, was the primary outcome. Results: The mean age of participants was 61.5 years, 57% (n = 91) were women, and 29.4% (n = 47) were non-white. At the end of treatment, acupuncture reduced insomnia severity by 8.3 points (95% CI 7.3-9.4), compared to 10.9 points (95% CI 9.8-12.0) for CBT-I, with CBT-I being the more effective treatment overall (2.6, 95% CI 1.1 – 4.1, P = 0.0007). Patients with mild insomnia were significantly more likely to respond to CBT-I than acupuncture (85% vs. 18%, p < 0.0001); however, patients with moderate to severe insomnia had similar response rates to CBT-I and acupuncture (75% vs. 66%, p = 0.26). Both groups had few mild adverse events and maintained improvements up to 20 weeks. Both groups also had similar improvement in quality of life in physical health (p = 0.46) and mental health (p = 0.44) during the study. Conclusions: While both acupuncture and CBT-I resulted in clinically meaningful and durable effects among cancer survivors with insomnia, CBT-I was more effective, especially among patients with mild insomnia symptoms. Patients and oncology clinicians can use these findings to inform their choice of insomnia treatment. Clinical trial information: NCT02356575.

Topic: 27. Thalassemias Background: Endocrinopathies remain the main complication of iron overload in Transfusion Dependent Thalassemia (TDT), hypogonadism, generally due to oxidative stress related pituitary damage, being the most frequent and often the earliest manifestation. A well-managed chelation therapy initiated during infancy reduces the risk of pubertal delay and hypogonadism. Few data about the current pubertal development of TDT patients receiving DFX are available in literature. Aims: The aim of this national study was to evaluate pubertal development and hypogonadism in TDT adolescents and young adults included in the French National Thalassemia Registry (NaThalY) who received oral chelation with DFX. Methods: This retrospective study concerned all TDT patients included in the national registry (HSCT recipients excluded) who received DFX for at least three years before puberty. Patients were born between 1997 (maximum age of 10 years when DFX was marketed in 2007) and 2009 (evaluable for puberty in 2022). Delayed puberty was defined as lack of testicular development at 14 years old in boys and absence of thelarche at 13 years old in girls. Simple delayed puberty was defined as puberty with a delayed onset but then proceeding normally without the need for any treatment to achieve full pubertal development and hypogonadism as insufficient synthesis of sexual hormones. Results: Fifty patients fulfilled inclusion criteria. Their median age at evaluation was 18 years (range 13-24) and 25 patients (50%) were male. The median duration of DFX chelation before puberty was 7 years (3 -12). 80% of patients were born in France. Regular transfusion regimen and chelation therapy were initiated early, at 1.3 years (0.2-4) and 3 years (1-6) respectively. The median age at puberty onset was 14 years in males and 12 years in females with a median age at first menstruation of 13.5 years (Table). Ten patients (20%) had pubertal development anomalies or hypogonadism. Six patients had simple pubertal delay (n=5) or a transient arrest of puberty with a spontaneous favorable outcome (n=1). One patient developed persistent primary amenorrhea despite a one-year estrogen-progestogen hormone therapy and three others had early secondary amenorrhea: one had a severe chronic iron overload, the second received hormone replacement therapy for a central and peripheral hypogonadism with premature ovarian insufficiency and the third patient had secondary amenorrhea just after a normal pregnancy (the only pregnancy registered in the cohort) currently under investigation. These 10 patients (median age of 20.5 years, range 16-24) had all a beta0/0 genotype and 7/10 experienced a history of severe iron overload (Serum ferritin > 2500 ug/L or liver iron concentration > 15 mg/g). Nonetheless the duration of DFX before puberty and the age at the start of chelation were similar to the other 40 patients with no delay in puberty onset. The final height reached in 35 patients was slightly reduced (-0.7 DS) with a median pubertal growth spurt (difference between final height and prepubertal height) of 18 cm in girls and 21.5 cm in boys. Summary/Conclusion: In adolescents and young adults starting chelation therapy early and receiving before puberty a median of 7 years of DFX, the residual frequency of pubertal anomalies or hypogonadism was of 20% half of them presenting a simple pubertal delay. A pubertal growth spurt was observed leading to a final height only mildly reduced. Specialized follow-up of puberty even in the absence of abnormality is mandatory in TDT children. In the next years the frequency of hypogonadism in young adults could be also assessed.Keywords: Iron overload, Blood transfusion, Iron chelation, beta thalassemia

Background: Differential expression analysis of (individual) genes is often used to study their roles in diseases. However, diseases such as cancer are a result of the combined effect of multiple genes. Gene products such as proteins seldom act in isolation, but instead constitute stable multi-protein complexes performing dedicated functions. Therefore, complexes aggregate the effect of individual genes (proteins) and can be used to gain a better understanding of cancer mechanisms. Here, we observe that complexes show considerable changes in their expression, in turn directed by the concerted action of transcription factors (TFs), across cancer conditions. We seek to gain novel insights into cancer mechanisms through a systematic analysis of complexes and their transcriptional regulation. Results: We integrated large-scale protein-interaction (PPI) and gene-expression datasets to identify complexes that exhibit significant changes in their expression across different conditions in cancer. We devised a log-linear model to relate these changes to the differential regulation of complexes by TFs. The application of our model on two case studies involving pancreatic and familial breast tumour conditions revealed: (i) complexes in core cellular processes, especially those responsible for maintaining genome stability and cell proliferation (e.g. DNA damage repair and cell cycle) show considerable changes in expression; (ii) these changes include decrease and countering increase for different sets of complexes indicative of compensatory mechanisms coming into play in tumours; and (iii) TFs work in cooperative and counteractive ways to regulate these mechanisms. Such aberrant complexes and their regulating TFs play vital roles in the initiation and progression of cancer.

Background: Primary stroke centers (PSCs) have been observed to have better stroke outcomes, and to have higher IV intravenous tissue-type plasminogen activator (IV tPA) utilization rates. Emergency medical services (EMS) began using routing protocols to direct suspected acute stroke patients to PSCs in the first decade of the century. We sought to determine the impact of county-level EMS stroke routing protocols on IV tPA utilization rates in California (CA). Methods: We extracted county-level patient discharge data from Office of Statewide Health Planning and Development database, an all-payer database including all hospitalizations in CA from 2006-2010. We used Diagnosis Related Group codes to identify all patients hospitalized for stroke, and patients who received a thrombolytic agent for stroke. We contacted EMS departments in CA and worked with the American Heart Association to determine which counties were utilizing EMS routing protocols and the year implemented. Using chi-square, we compared tPA usage in routing counties vs. non-routing counties. Among counties that implemented routing protocols, we compared tPA usage 1 year pre- and post-routing protocol implementation. Results: Of 58 counties in CA, 19 were identified to have implemented acute stroke EMS routing protocols in years 2006 and 2010. Across this 5 year period, use of tPA among counties without a concurrent EMS routing protocol was 2.1% [1663/79148], whereas use of tPA among counties with a concurrent EMS routing protocol was 3.1% [4853/154962] (p&lt;0.001). Among the 39 counties that have not yet implemented EMS routing protocols, tPA use increased from 1.3% to 3.1% from 2006-2010. In the same time period, the 19 counties with EMS routing protocols showed tPA use increased from 2.3% to 4.4%. Further analysis was performed for counties whose data for tPA usage 1 year pre- and post-routing were available: San Francisco County (2007); Alameda, Kern, San Mateo Counties (2008); Los Angeles, Orange, Sacramento, San Diego Counties (2009). Counties that implemented EMS acute stroke routing protocols increased tPA rates usage from 2.6% in the year before routing to 4.0% in the year after routing (p&lt;0.001). Conclusions: Counties with EMS protocols to route acute stroke patients to PSCs have higher IV tPA rates than non-routing counties. Routing counties are also seen to increase their rate of IV tPA after beginning implementation of routing protocols. To continue to improve acute stroke care through the usage of IV tPA, current non-routing counties should consider implementing routing protocols to transport acute stroke patients to PSCs.

Background Timely and efficient reperfusion is associated with better outcome from acute cerebral ischemia. The predictors of procedural success in patients treated with multimodal mechanical device strategies (Merci ± Penumbra ± angioplasty and stenting) have not been well delineated. Methods In a prospectively maintained database, we analyzed consecutive patients with acute ICA and M1 occlusions treated with endovascular recanalization following multimodal MRI. We investigated the pretreatment clinical and imaging factors affecting three procedural outcomes: number of passes, single device-type therapy, and presence of SAH after the procedure. We also sought to determine the relationship between these procedural variables with substantial recanalization (TICI≥2b) and clinical outcome. Results Among 105 patients meeting study entry criteria, mean age was 66.6 (±17.8), 65% were female and 34% had history of atrial fibrillation. The median pretreatment NIHSS was 18 (range 2-31), mean baseline DWI volume was 30.6cc (SD±35.1), and mean time to groin puncture was 412min (SD±207.6). The median number of mechanical device passes was 2 (range 0-8). 73 (70%) patients were treated with a single device. IV tPA was used in 43 patients (41%). Substantial recanalization occurred in 43 patients (41%). In the final binary logistic regression multivariate analysis, among all baseline clinical and imaging variables, history of atrial fibrillation was the most significant factor associated with a single device therapy (OR 0.249; p=0.024). Age, gender, baseline DWI volumes, arterial occlusion site and time to recanalization did not correlate with the number of attempts, single or multiple device usage, or presence of SAH after the procedure. None of the procedural or baseline variables correlated with recanalization rates. The strongest predictors of poor outcome (mRS≥3 at discharge) were high baseline NIHSS (OR 0.87; p&lt;0.001) and presence of SAH after the procedure (OR 0.05; p=0.001). However, the presence of SAH did not correlate with the number of attempts or devices used. Conclusions: A history of atrial fibrillation predicts single device usage in mechanical thrombectomy for acute ischemic stroke treatment. This is likely due to the fibrin-rich histological composition of the clot. In contrast to prior studies involving a single type of device, increased number of passes with multiple different mechanical devices was not associated with lower recanalization rates and did not worsen clinical outcome.

pcot2 is an R-package for the analysis of groups of genes in microarray experiments. It utilizes inter-gene correlation information to detect significant alterations in the activities of gene sets. Incorporating additional (usually functional) information into the data analysis process allows gene interactions to be investigated in a statistical framework. One of the reasons that gene set analysis is becoming important is that it is suitable for detecting small coordinated changes in expression of groups of genes which are functionally related, which may not be considered significant in a single gene analysis. This vignette gives a tutorial-style introduction to the functions in the pcot2 package. These functions are used for testing and visualizing changes in expression activity for groups of genes.

B172 Recently we reported that high proliferation in colorectal and gastric cancers is associated with improved clinical outcome (in terms of increased disease-free survival, relative to less-proliferative tumours), based on an expression-based gene proliferation signature [1]. The reverse relationship (i.e., high proliferation associated with poor outcome) was observed in multiple microarray data sets from a number of non-gastrointestinal cancers, including breast, glioma and lymphoma, using the same gene-based assessment of proliferation. Earlier work in our laboratory also identified a gene signature for colorectal cancer prognosis prediction in which higher levels of expression of immune response related genes were associated with increased disease free survival [2].
 Here we use newly developed bioinformatic methodology to investigate the biology underlying this somewhat counterintuitive relationship between proliferation and prognosis in colorectal and gastric cancer, in the context of an expression-based assessment of both proliferation level and immune response for each tumor. This approach is based on identifying sets of correlated genes which undergo statistically significant alterations in expression between prognosis, proliferation, and/or immune response classes in colorectal and gastric cancer, and contrasting these relationships with those observed in breast cancer, for which there is both a large amount of high quality publicly available microarray data, as well as a widely accepted association between high proliferation and poor prognosis.
 This approach has revealed that while highly proliferative tumors express immune response related genes in a similar pattern across colorectal, gastric and breast cancers, certain genes appear to be down-regulated in highly proliferative breast cancers (relative to low proliferation tumors) that remain unchanged in colorectal and gastric tumors. Our conclusion is that subtle differences in the host immune response underly the differences in the relationship between proliferation and prognosis in colorectal, gastric and breast cancers.
 1. Lin YH, et al. A proliferation gene signature can predict clinical outcome of multiple cancers. Abstract LB-264, 2007 AACR Annual Meeting, Los Angeles, CA.
 2. Lin, YH, et al. Multiple gene expression classifiers from different array platforms predict poor prognosis of colorectal cancer., Clin Cancer Res, 13, 2 Pt 1, 2007.

<b>Summary</b><br> This metadata record provides details of the data supporting the claims of the related manuscript: “Effects of systemic inflammation on relapse in early breast cancer”. The data consist of a single data spreadsheet in Stata and open format, and a supporting data dictionary in <b>.txt</b> format. The related study aimed to assess whether or not elevated serum inflammatory biomarkers (C-Reactive protein [CRP], interleukin-6 [IL-6], and serum amyloid A [SAA]) and/or the presence of a high-risk IL-6 promoter genotype were associated with recurrence of hormone receptor positive (HR+) early breast cancer.<br> <b> </b> <b>Data access</b> The data generated and/or analysed during the related study is openly available as part of this metadata record. The data are as follows:- <b>WABC Case Control Deidentified Dataset.xlsx</b> – De-identified dataset for WABC-II Case Control population with serum and genomic biomarkers as well as outcomes.- <b>WABC Case Control Deidentified Dataset.dta</b> – Stata version of the above.- <b>WABC_data_dictionary.txt</b> – Data dictionary to support the above files. <b><br></b><b>Name of Institutional Review Board or ethics committee that approved the study</b> This study was approved by the Institutional Review Board of the University of Pennsylvania and all study procedures were conducted according to the institution’s code of ethics.

This project concentrates on developing a nonparametric Empirical Bayes (EB) method on predicting patients' survival times based on the gene expression profiles. Since the number of redundant variables is large in microarray data, the traditional model selection criteria, such as AIC, BIC etc., do not work properly. The nonparametric EB method is used to find a new threshold based on an estimated latent distribution. This not only reduces the dimensions of the model but also increases the predictive accuracy of the selected model. We estimate a mixture model of chi-squared distribution with censored data via EM algorithm. The mathematical derivation and computational implementation are presented. A diffuse large B-cell lymphoma (DLBCL) dataset is used as an illustrating example in this project. Evaluation on an independent test data and two simulations have been done to demonstrate the methodology. The nonparametric EB method is a potentially powerful tool for detecting the effect of individual genes on survival times and for finding the best possible model in the a large-scaled dataset.

Abstract Background The accurate detection of copy number alterations from the analysis of circulating cell free tumour DNA (ctDNA) in blood is essential to realising the potential of liquid biopsies. However, currently available approaches require a large number of plasma samples from healthy individuals, sequenced using the same platform and protocols to act as a reference panel. Obtaining this reference panel can be challenging, prohibitively expensive and limits the ability to migrate to improved sequencing platforms and improved protocols. Methods We developed qCNV and sCNA-seq, two distinct tools that together provide a new approach for profiling somatic copy number alterations (sCNA) through the analysis of cell free DNA (cfDNA) without a reference panel. Our approach was designed to identify sCNA from cfDNA through the analysis of a single plasma sample and a matched normal DNA sample -both of which can be obtained from the same blood draw. qCNV is an efficient method for extracting read-depth from BAM files and sCNA-seq is a method that uses a probabilistic model of read depth to infer the copy number segmentation of the tumour. We compared the results from our pipeline to the established copy number profile of a cell-line, as well as the results from the plasma-Seq analysis of cfDNA-like mixtures and real, clinical data-sets. Results With a single, unmatched, germline reference sample, our pipeline recapitulated the known copy number profile of a cell-line and demonstrated similar results to those obtained from plasma-Seq. With less than 1X genome coverage, our approach identified clinically relevant sCNA in samples with as little as 20 % tumour DNA. When applied to plasma samples from cancer patients, our pipeline identified clinically significant mutations. Conclusions These results show it is possible to identify therapeutically-relevant copy number mutations from plasma samples without the need to generate a reference panel from a large number of healthy individuals. Together with the range of sequencing platforms supported by our qCNV+sCNA-Seq pipeline, as well as the Galaxy implementation of this solution, this pipeline makes cfDNA profiling more accessible and makes it easier to identify sCNA from the plasma of cancer patients.