Search

Background Comprehensive molecular profiling has revealed somatic variations in cancer at genomic, epigenomic, transcriptomic, and proteomic levels. The accumulating data has shown clearly that molecular phenotypes of cancer are complex and influenced by a multitude of factors. Conventional unsupervised clustering applied to a large patient population is inevitably driven by the dominant variation from major factors such as cell-of-origin or histology. Translation of these data into clinical relevance requires more effective extraction of information directly associated with patient outcome. Methods Drawing from ideas in supervised text classification, we developed survClust, an outcome-weighted clustering algorithm for integrative molecular stratification focusing on patient survival. survClust was performed on 18 cancer types across multiple data modalities including somatic mutation, DNA copy number, DNA methylation, and mRNA, miRNA, and protein expression from the Cancer Genome Atlas study to identify novel prognostic subtypes. Results Our analysis identified the prognostic role of high tumor mutation burden with concurrently high CD8 T cell immune marker expression and the aggressive clinical behavior associated with CDKN2A deletion across cancer types. Visualization of somatic alterations, at a genome-wide scale (total mutation burden, mutational signature, fraction genome altered) and at the individual gene level, using circomap further revealed indolent versus aggressive subgroups in a pan-cancer setting. Conclusions Our analysis has revealed prognostic molecular subtypes not previously identified by unsupervised clustering. The algorithm and tools we developed have direct utility toward patient stratification based on tumor genomics to inform clinical decision-making. The survClust software tool is available at https://github.com/arorarshi/survClust.

Autophagy promotes protein degradation, and therefore has been proposed to maintain amino acid pools to sustain protein synthesis during metabolic stress. To date, how autophagy influences the protein synthesis landscape in mammalian cells remains unclear. Here, we utilize ribosome profiling to delineate the effects of genetic ablation of the autophagy regulator, ATG12, on translational control. In mammalian cells, genetic loss of autophagy does not impact global rates of cap dependent translation, even under starvation conditions. Instead, autophagy supports the translation of a subset of mRNAs enriched for cell cycle control and DNA damage repair. In particular, we demonstrate that autophagy enables the translation of the DNA damage repair protein BRCA2, which is functionally required to attenuate DNA damage and promote cell survival in response to PARP inhibition. Overall, our findings illuminate that autophagy impacts protein translation and shapes the protein landscape., Goldsmith et al. employ ribosome profiling to dissect how deletion of the essential autophagy component Atg12 impacts mRNA translation. They detect changes in the translation of mRNAs involved in DNA repair, centrosome clustering and cell cycle control and specifically focus on how loss of autophagy causes reduced production of BRCA2 and increased DNA damage.

Purpose To determine the usefulness of a comprehensive, targeted-capture next-generation sequencing (NGS) assay for the clinical management of children undergoing enucleation for retinoblastoma. Design Cohort study. Participants Thirty-two children with retinoblastoma. Methods We performed targeted NGS using the UCSF500 Cancer Panel (University of California, San Francisco, San Francisco, CA) on formalin-fixed, paraffin-embedded tumor tissue along with constitutional DNA isolated from peripheral blood, buccal swab, or uninvolved optic nerve. Peripheral blood samples were also sent to a commercial laboratory for germline RB1 mutation testing. Main Outcome Measures Presence or absence of germline RB1 mutation or deletion, tumor genetic profile, and association of genetic alterations with clinicopathologic features. Results Germline mutation or deletion of the RB1 gene was identified in all children with bilateral retinoblastoma (n = 12), and these NGS results were 100% concordant with commercial germline RB1 mutation analysis. In tumor tissue tested with NGS, biallelic inactivation of RB1 was identified in 28 tumors and focal MYCN amplification was identified in 4 tumors (2 with wild-type RB1 and 2 with biallelic RB1 inactivation). Additional likely pathogenic alterations beyond RB1 were identified in 13 tumors (41%), several of which have not been reported previously in retinoblastoma. These included focal amplifications of MDM4 and RAF1, as well as damaging mutations involving BCOR, ARID1A, MGA, FAT1, and ATRX. The presence of additional likely pathogenetic mutations beyond RB1 inactivation was associated with aggressive histopathologic features, including higher histologic grade and anaplasia, and also with both unilateral and sporadic disease. Conclusions Comprehensive NGS analysis reliably detects relevant mutations, amplifications, and chromosomal copy number changes in retinoblastoma. The presence of genetic alterations beyond RB1 inactivation correlates with aggressive histopathologic features.

Glucocorticoids (GCs) are a central component of therapy for patients with T cell acute lymphoblastic leukemia (T-ALL), and although resistance to GCs is a strong negative prognostic indicator in T-ALL, the mechanisms of GC resistance remain poorly understood. Using diagnostic samples from patients enrolled in the frontline Children's Oncology Group (COG) T-ALL clinical trial AALL1231, we demonstrated that one-third of primary T-ALLs were resistant to GCs when cells were cultured in the presence of IL-7, a cytokine that is critical for normal T cell function and that plays a well-established role in leukemogenesis. We demonstrated that in these T-ALLs and in distinct populations of normal developing thymocytes, GCs paradoxically induced their own resistance by promoting upregulation of IL-7 receptor (IL-7R) expression. In the presence of IL-7, this augmented downstream signal transduction, resulting in increased STAT5 transcriptional output and upregulation of the prosurvival protein BCL-2. Taken together, we showed that IL-7 mediates an intrinsic and physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALLs and is reversible with targeted inhibition of the IL-7R/JAK/STAT5/BCL-2 axis.

KRAS mutations cause a quarter of cancer mortality and currently are not sensitive to any targeted, FDA-approved agents. Several inhibitors of the MAPK pathway are FDA approved but exhibit low clinical tolerability at doses needed to adequately extinguish KRAS signaling. We discovered an avidity for ferrous iron (Fe2+) induced by and dependent on oncogenic KRAS signaling. We leveraged anFDA-approved MEK inhibitor to produce a prototypical Ferrous Iron–Activatable Drug Conjugate (FeADC) and with this novel agent achieved MAPK blockade in tumor cells while sparing normal tissues. These improvements allowed sustainable, effective treatment of tumor bearing animals with superior tolerability. Iron-activated therapeutics are unknown outside of antiparasitic therapy, but may hold significant promise for the treatment of KRAS-driven solid tumors.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution., Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors utilize DNA methylation based subgroups in JMML to predict clinical outcome.

Many complex diseases are caused by a variety of both genetic and environmental factors acting in conjunction. To help understand these relationships, nonparametric methods that use aggregate learn...

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare and highly aggressive myeloid malignancy of early childhood. Approximately 95% of patients have at least one mutation that leads to hyperactive RAS signaling. Both the presence of multiple mutations at diagnosis as well as altered DNA methylation are associated with a poor prognosis. Whether altered DNA methylation leads to the acquisition of additional genetic mutations or is a secondary event to genetic mutations is unknown. In this study, we analyzed a total of 33 newborn blood screening (NBS) cards from children who went on to develop JMML later in childhood. Using next generation sequencing to detect both genetic mutations and DNA methylation status we sought to determine the sequence of events that lead to the development of JMML. Patients and Methods We identified 33 patients that were born in the state of California from 1990 to 2017 who were confirmed to have JMML and were previously consented to participate in a JMML tissue bank study. Guthrie cards from these 33 patients as well as 12 healthy controls were obtained from the California Biobank Program. DNA samples (20 ng) were sequenced for mutations using a custom amplicon-based sequencing approach (Paragon Genomics, Hayward, CA) targeting 26 genes that are recurrently mutated in JMML. For DNA methylation profiling, 300ng of genomic DNA was processed for bisulfite conversion using the TrueMethyl oxBS Module (Tecan Genomics Inc., Redwood City, CA.) according to manufacturer's guidelines and next generetation sequencing (NGS) libraries were prepared by targeting 3000 CpG loci using custom probes for Targeted Methyl-Seq assay (Tecan Genomics Inc., Redwood City, CA). Sequence reads were trimmed using Cutadapt to remove adapters and methylation status called using Bismark. Samples were classified into one of three methylation subgroups: Low, Intermediate, or High using 1386 probes according to an international consensus definition. Results At diagnosis of JMML, somatic mutations were identified in 32 of 33 patients. Of the 32 patients with a somatic mutation present upon diagnosis, the same mutations were found in NBS cards in 13 (41%) patients using a mutant allele fraction (MAF) cut-off of 1%. Clonal mutations (MAF >15%) were found in 9 of 32 (28%) patients. Patients who had a somatic mutation detected at birth were significantly younger at diagnosis with a median age of 7.1 months (range 2.5-91.6 months) compared to patients who had none (19.8 months; p-value = 0.042). However, no difference was observed in overall or event-free survival for patients with or without somatic mutations at birth. Methylation profiling classified all NBS cards as having "low" DNA methylation using an international, consensus definition (Figure 1A). Of the 33 patients, ten also had DNA methylation profiling performed on their diagnostic JMML sample which were classified as low (LM), intermediate (IM) or high methylation (HM) (Figure 1B). Amongst patients with both NBS and JMML methylation data available, 5 had mutations present in the NBS card and all 5 had lower methylation (β values) in their NBS card compared to their JMML sample (mean 0.38 vs 0.29, p-value = 0.002). Discussion We identified somatic Ras pathway mutations at birth in 13/32 (41%) of newborns that developed JMML later in their childhood. We found that patients with a somatic mutation detected at birth were significantly younger at diagnosis compared to patients who had no mutations at birth. DNA methylation profiling of NBS cards from children who went on to develop JMML revealed methylation signatures that were similar to normal age-matched controls. All newborn cards were classified as low methylation at birth. Specifically, amongst patients who had genetic mutations present at birth, methylation changes that were eventually detected in JMML samples were not present in the NBS cards. Therefore, we conclude that aberrant DNA methylation is a secondary event to genetic changes in JMML. Figure 1: Panel A: Methylation profiling of JMML NBS and control NBS samples, classified according to the international JMML methylation consensus signature. Panel B: Methylation profiling of paired NBS and diagnosis samples from JMML patients. The star indicates the NBS methylation profile. Heatmaps (A and B) show the beta values of 1386 CpG loci used for methylation classification. Figure 1 Disclosures Loh: Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

10002 Background: The heterogeneity of T-ALL has hindered biomarker identification and limited biology-based risk stratification. Historically, minimal residual disease (MRD) has been the strongest predictor of poor outcomes. However, stratification by MRD does not allow for risk-adapted therapy early in treatment, which may induce deeper remissions and decrease risk of relapse. We hypothesized that gene expression profiling at diagnosis may have prognostic value in identifying high risk patients. Methods: We analyzed RNA-seq data from 189 diagnostic samples from the Children’s Oncology Group (COG) AALL0434 trial. Using leave-one-out cross-validation, we identified a set of genes that optimally differentiated MRD+ and MRD- samples. We then derived a risk score (RS) that indicates a probability of being MRD+ for a given gene expression pattern. Finally, we validated this model in an independent cohort of COG AALL1231 samples. Results: The AALL0434 early T-cell precursor (ETP) samples (n = 19), which have high rates of MRD+, had the highest RS, with an average of 81.3 (SD 18.7), versus 24.9 (SD 22.7) for non-ETPs (n = 146). Intriguingly, non-ETPs with RS > 50 had a gene expression pattern that mirrored ETPs and was distinct from the remaining non-ETPs. In this RS > 50 non-ETP cohort, 80% were MRD+, versus 20% of the < 50 cohort (p < 0.0001). When applied to 31 diagnostic non-ETP samples from COG AALL1231, 57% of the RS > 50 cohort were MRD+, versus 17% of the RS < 50 cohort (p = 0.05). Importantly, AALL0434 used prednisone during induction, while AALL1231 used dexamethasone, indicating that the predictive value is independent of the induction steroid. Finally, we converted our model to the customizable Nanostring nCounter platform by analyzing 96 AALL0434 samples on the Nanostring assay. The Nanostring data closely recapitulated the RNA-seq data, with a tight correlation between the resulting RS (concordance correlation coefficient = 0.91). Conclusions: We have developed a gene expression classifier that differentiates a subset of non-ETP T-ALLs with an ETP-like gene expression pattern and a high risk of MRD+, and have adapted the classifier to a clinically tractable targeted platform. Identification of this high-risk subset at diagnosis has the potential to facilitate risk-adapted trials to evaluate the utility of novel or more intensive therapies aimed at improving clinical outcomes.

Objective The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. Materials and methods Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109. Results UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers. Conclusion We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.

BACKGROUND Previous studies of solid tumors have been restricted in their ability to map how heterogeneous cell populations evolved within the tumor in three-dimensional (3D) space due to insufficient sampling, typically one sample per tumor, and a lack of knowledge of where within the tumor the sample was obtained. Knowledge of the extensivity of heterogeneity and how it is spatially distributed is crucial for assessing whether a therapeutic target is truly tumor-wide, and for exploring how mutations relate to heterogeneity in the local microenvironment. METHODS We developed a novel platform to integrate and visualize in 3D multi-omics data generated from each of 8–10 spatially mapped samples per tumor. Together, the 171 samples collected using this approach from 16 adult diffuse glioma at diagnosis and recurrence form a novel resource – the 3D Glioma Atlas. RESULTS By maximally sampling the tumor geography without excluding samples based on low cancer cell fraction (CCF), we identify a subpopulation of glioblastoma with pervasively lower CCF likely excluded by other atlases, such as the TCGA, that used stringent CCF cutoffs. Exome sequencing of 3D-mapped samples from lower-grade tumors revealed that clonal expansions are typically spatially segregated, implying minimal tumor-wide intermixing of genetically heterogenous cells. Heterogeneity is less spatially segregated for faster-growing high-grade tumors, suggesting that cell populations expand in these tumors differently. Recurrent low-grade tumors have greater intratumoral mutational heterogeneity than newly diagnosed tumors, though this did not translate into greater dissimilarity in gene expression profiles for recurrent tumors, suggesting minimal functional impact of this additional mutational diversity on gene expression. CONCLUSIONS The delineation of spatial patterns of heterogeneity that our work provides enables more informed interpretation of biopsies and greater insight into the factors shaping intratumoral variation of gene expression patterns. Ongoing work is exploring the spatial patterning of amplification events and the tumor microenvironment.

Author(s): Afshar, Armin R; Damato, Bertil E; Stewart, Jay M; Zablotska, Lydia B; Roy, Ritu; Olshen, Adam B; Joseph, Nancy M; Bastian, Boris C | Abstract: PurposeTo clinically use the UCSF500, a pancancer, next-generation sequencing assay in uveal melanoma (UM) and to correlate results with gene expression profiling (GEP) and predictive factors for metastasis.MethodsCohort study. Tumor samples of adult UM patients were analyzed with the UCSF500 and GEP. Main outcomes were copy number changes in chromosomes 1, 3, 6, and 8 and mutations in GNAQ, GNA11, SF3B1, EIF1AX, BAP1, SRSF2, U2AF1, and PLCB4. Chromosome 3 loss (a metastasis predictor) was tested for correlation with GEP class, tumor characteristics (largest basal diameter, thickness, ciliary body involvement, and extraocular extension), and histology (presence of epithelioid cells, closed loops, and mitotic count).ResultsThe 62 patients had a mean age of 59 years (range, 24-89 years). Chromosome 3 loss was detected in 30 patients and was associated with larger basal tumor diameter (Wilcoxon rank sum test, P = 0.015), greater thickness (Wilcoxon rank sum test, P = 0.016) and tumor, node, metastasis stage (Fisher test, P = 0.006), epithelioid cytology (Fisher test, P l 0.001), BAP1 mutation (Fisher test, P l 0.001), and chromosome 8q gain (Fisher test, P l 0.001). Class 2 tumors were much more likely to have chromosome 3 loss than class 1 (odds ratio, 121; P l 0.001). Eleven patients developed metastatic UM, of which five died during the study. All metastatic cases had chromosome 3 loss, 8 gain, BAP1 mutation, and class 2 GEP. Five class 1 tumors had chromosome 3 loss.ConclusionsUCSF500 detects chromosomal copy number changes and missense mutations that correlate strongly with metastasis predictors, including GEP.Translational relevanceNext-generation sequencing of UM should enhance survival prognostication.

Non-response to anti-PD-1 antibodies is characterized by a “cold” tumor microenvironment lacking “exhausted” CD8+ T cells. We report on the first prospective Phase 2 trial of intratumoral plasmid IL-12 and pembrolizumab in advanced melanoma with “exhausted” T-cell (peCTL) poor tumors that had a low pre-test probability of response to anti-PD-1 Ab monotherapy. Patients had a 43% objective response rate (n=23, RECIST 1.1) with 38% complete responses. Interrogation of samples demonstrated that, while intratumoral IL-12 triggered a rapid increase in immune signaling and licensing of the tumor microenvironment, the compensatory adaptive resistance blunted clinical responses. However, by disrupting the PD-1/PD-L1 axis with pembrolizumab, the IL-12/IFN-γ feed-forward cycle was sustained, driving intratumoral cross-presenting DC subsets with increased TIL and TCR clonality and, ultimately, systemic cellular immune responses. Thus, combination of intratumoral plasmid IL-12 with pembrolizumab transformed poorly immunogenic lesions into effective in situ vaccines, attaining clinical activity in low peCTL tumors.

Mutational hotspots indicate selective pressure across a population of tumor samples, but their prevalence within and across cancer types is incompletely characterized. An approach to detect significantly mutated residues, rather than methods that identify recurrently mutated genes, may uncover new biologically and therapeutically relevant driver mutations. Here we developed a statistical algorithm to identify recurrently mutated residues in tumour samples. We applied the algorithm to 11,119 human tumors, spanning 41 cancer types, and identified 470 hotspot somatic substitutions in 275 genes. We find that half of all human tumors possess one or more mutational hotspots with widespread lineage-, position-, and mutant allele-specific differences, many of which are likely functional. In total, 243 hotspots were novel and appeared to affect a broad spectrum of molecular function, including hotspots at paralogous residues of Ras-related small GTPases RAC1 and RRAS2. Redefining hotspots at mutant amino acid resolution will help elucidate the allele-specific differences in their function and could have important therapeutic implications.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 and CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and therefore be candidates for experimental therapies. In addition, there have been few other molecular pathways identified aside from the Ras/MAPK pathway to serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia in order to expand our knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, gene splicing, the polycomb repressive complex 2 (PRC2) and transcription. Importantly, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.

Introduction: Juvenile myelomonocytic leukemia is a heterogenous disease of early childhood that has both myelodysplastic and myeloproliferative properties. Outcomes range from spontaneous resolution with little to no therapy in some patients while others relapse even after hematopoietic stem cell transplantation. However, there is no single biomarker reliably capable of predicting outcome. Patients and Methods: Our three groups have recently published data regarding the predictive capacity of DNA methylation profiling using the Illumina Infinium Methylation 450k platform. Methylation data along with clinical annotation from 256 JMML patients (126 EWOG/MDS, 93 Japanese and 37 USA) were analyzed and a consensus algorithm to determine DNA methylation clustering was agreed upon. A technical validation of two clinically oriented DNA methylation approaches was undertaken using DNA from 32 patients (12 EWOG/MDS, 5 Japan and 15 USA) included in the previous publications. A biological validation using DNA from additional 48 JMML patients (9 EWOG/MDS, 19 Japan and 20 USA) that were not previously tested was also undertaken to independently validate the DNA methylation subgroups. All 80 patients from the technical and biological cohorts were analyzed for genetic mutations using a custom 25-gene panel next-generation-sequencing (NGS) approach. Informed consent to use of patient material was obtained in accordance with the Declaration of Helsinki and approved by the local institutional review boards. Results: Unsupervised hierarchical consensus clustering of 256 JMML patients using the 5000 CpG sites with the highest standard deviation separated patients into three distinct methylation subgroups using the 450k data. Based on the mean beta-value detected, the subgroups were designated as low methylation (LM), intermediate methylation (IM) and high methylation (HM). Strikingly, when comparing our new DNA methylation subgroup assignments to the published 450k DNA methylation subgroups, only 11 patients (3.86%) were now re-classified into a different methylation subgroup (Figure 1). Next two approaches were taken with the intent of clinical grade implementation of DNA methylation testing. In one approach at EWOG/MDS, a multiclass classification model (XGBoost) was developed based on the 5000 CpGs and tested by classifying the validation cohort into the respective methylation subgroups. When analyzing the relative contribution of each CpG to the model performance (Model Gain), we identified 10 CpG sites which contributed most to the model. Illumina Infinium 850k array testing using XGBoost of the technical cohort provided an accuracy of 90.6% resulting in misclassification of 3/32 patients as compared to the original 450k based designation of methylation subgroups. In the USA, an amplicon based, bisulfite treated, NGS approach (Methyl-NGS) using primers to sequence the top 3000 CpG sites from the meta-analysis was tested in the technical and biological validation cohorts. Using a standard deviation of 0.25 of the most variable probes to determine the methylation clusters, there was 100% concordance between the 450k and the Methyl-NGS designation of methylation subgroups. Conclusions: In this single largest collection of JMML data ever presented, we demonstrate the reproducibility of DNA methylation as a predictive biomarker of outcome across continents, platforms and different patient cohorts. The DNA methylation consensus subgroups generated from 256 JMML patients were associated with previously recognized variables such as age, platelet count, fetal hemoglobin status and the number of genetic alterations at diagnosis. However, DNA methylation cluster designation provided additional prognostic information, in particular in young patients or patients with monosomy 7 where fetal hemoglobin is not interpretable and clarified that not all patients with PTPN11, KRAS or NF1 are classified as IM or HM as might be expected based on genetics alone. Clinical grade implementation of DNA methylation testing using either Infinium based XGBoost or Methyl-NGS both provided a high degree of reproducibility with the original 450k data. Combining DNA methylation with previously recognized variables allows for a robust risk-stratification algorithm that is now ready to be implemented in clinics around the world and integrated into clinical trials. Disclosures Loh: Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Niemeyer:Celgene: Consultancy. Lipka:InfectoPharm GmbH: Employment.

Hodgkin lymphoma (HL) is the most common malignancy affecting adolescents and young adults. Treatment with a combination of chemotherapy and radiation results in cure rates of >90%. However, radiation therapy causes significant late effects and avoiding radiation entirely for patients who respond to chemotherapy is an accepted strategy. Since 2011, 28 consecutive patients diagnosed with classic HL have been treated with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) for 4 to 6 cycles. Patients who achieved a complete metabolic response (CMR) as assessed by [F] fluorodeoxyglucose positron emission tomography by the end of chemotherapy did not receive radiation. Among the 27 evaluable patients, 26/27 (96.2%) achieved a CMR with ABVD alone with 24/27 (88.9%) having achieved a CMR after 2 cycles. Event-free survival at 5 years is 90.5% and overall survival is 100% with a median follow-up time of 22.4 and 22.1 months, respectively. Treating pediatric and young adult HL patients with ABVD alone results in CMRs in >95% of patients. Patients who were refractory to ABVD or relapsed after treatment eventually achieved remission with a combination of standard and novel salvage therapies. This regimen demonstrates the feasibility of avoiding upfront radiation in newly diagnosed pediatric HL patients.

Desmoplastic melanoma is an uncommon variant of melanoma with sarcomatous histology, distinct clinical behavior and unknown pathogenesis. We performed low-coverage genome and high-coverage exome sequencing of 20 desmoplastic melanomas, followed by targeted sequencing of 293 genes in a validation cohort of 42 cases. A high mutation burden (median of 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers. Mutation patterns strongly implicate ultraviolet radiation as the dominant mutagen, indicating a superficially located cell of origin. Newly identified alterations included recurrent promoter mutations of NFKBIE, encoding NF-κB inhibitor ɛ (IκBɛ), in 14.5% of samples. Common oncogenic mutations in melanomas, in particular in BRAF (encoding p.Val600Glu) and NRAS (encoding p.Gln61Lys or p.Gln61Arg), were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS and PIK3CA, some of which are candidates for targeted therapies.

SummaryThe evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution.

Genomic studies have revealed significant branching heterogeneity in cancer. Studies of resistance to tyrosine kinase inhibitor therapy have not fully reflected this heterogeneity because resistance in individual patients has been ascribed to largely mutually exclusive on-target or off-target mechanisms in which tumors either retain dependency on the target oncogene or subvert it through a parallel pathway. Using targeted sequencing from single cells and colonies from patient samples, we demonstrate tremendous clonal diversity in the majority of acute myeloid leukemia (AML) patients with activating FLT3 internal tandem duplication mutations at the time of acquired resistance to the FLT3 inhibitor quizartinib. These findings establish that clinical resistance to quizartinib is highly complex and reflects the underlying clonal heterogeneity of AML.

Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.

10033 Background: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease, which has largely precluded the use of genetic mutations for risk stratification. We hypothesized that despite this heterogeneity, diverse T-ALLs may have functional similarities that underlie patterns of chemotherapy sensitivity. Methods: We used flow cytometry to evaluate in vitro dexamethasone (DEX) sensitivity and baseline expression of signal transduction effectors and BCL2-family proteins in 68 fresh diagnostic T-ALL samples from patients enrolled on the Children’s Oncology Group (COG) trial AALL1231. We also performed RNA-sequencing (RNA-seq) on 40 AALL1231 samples and used hierarchical clustering and linear regression to analyze these and published T-ALL RNA-seq data from COG AALL0434. Comparisons between groups were made using t-tests and Fisher’s exact tests. Results: Of the proteins analyzed, only high BCL2 expression was significantly associated with increased in vitro DEX resistance (p = 0.002). Hierarchical clustering of the AALL1231 RNA-seq data identified two distinct clusters. Cluster 1 was associated with significantly higher BCL2 transcript expression (p = 0.0002) and in vitro DEX resistance (p = 0.04) relative to cluster 2. We defined a gene set consisting of the top 210 differentially expressed genes between these clusters and applied this gene set to the COG AALL0434 cohort. In this analysis, the early T-cell precursor (ETP) and near-ETP samples clustered together (p < 0.0001) in cluster 1 along with 39 of 146 non-ETP samples. Not only did these cluster 1 non-ETP samples have significantly higher BCL2 transcript expression relative to the non-ETP samples in cluster 2 (p < 0.0001), but 54% of these non-ETP samples were minimal residual disease (MRD) positive (≥0.01%) at the end of induction, as opposed to only 16% of the non-ETP samples in cluster 2 (p < 0.0001). Conclusions: Gene expression profiling identifies non-ETP T-ALLs that cluster with ETP/near-ETP T-ALLs and have significantly higher BCL2 expression and increased rates of post-induction MRD.

Background We investigated circulating levels of individual soluble urokinase plasminogen activation receptor (suPAR) forms to determine if specific circulating fragments of suPAR (II-III) and (I) can better serve as clinical biomarkers for focal segmental glomerulosclerosis (FSGS) and the risk of recurrence after transplantation. Materials and methods Serum levels of intact suPAR and its cleaved forms were measured with two assays, ELISA and TR-FIA. Results suPAR levels in healthy controls were significantly lower than those who had glomerular diseases but were not significantly different between FSGS patients and glomerular controls. Intact suPAR (I-II-III) levels were noted to be elevated in glomerular diseases including FSGS. uPAR fragment (I) levels measured with the TR-FIA 4 assay were significantly higher in FSGS (695.4 + 91.29 pMol/L) than glomerular controls (239.1 + 40.45 pMol/L, P = 0.001). However, suPAR(I) levels were not significantly different between recurrent FSGS and nonrecurrent FSGS patients. Conclusion Our analysis of suPAR using the ELISA assay used in all previous studies does not appear to be a useful marker for FSGS nor serve as a predictor for its recurrence after transplantation. The TR-FIA assay results suggest that uPAR(I) is a potential biomarker for FSGS but not of its recurrence.

Gene regulation during cell-cycle progression is an intricately choreographed process, ensuring accurate DNA replication and division. However, the translational landscape of gene expression underlying cell-cycle progression remains largely unknown. Employing genome-wide ribosome profiling, we uncover widespread translational regulation of hundreds of mRNAs serving as an unexpected mechanism for gene regulation underlying cell-cycle progression. A striking example is the S phase translational regulation of RICTOR, which is associated with cell cycle-dependent activation of mammalian target of rapamycin complex 2 (mTORC2) signaling and accurate cell-cycle progression. We further identified unappreciated coordination in translational control of mRNAs within molecular complexes dedicated to cell-cycle progression, lipid metabolism, and genome integrity. This includes the majority of mRNAs comprising the cohesin and condensin complexes responsible for maintaining genome organization, which are coordinately translated during specific cell cycle phases via their 5' UTRs. Our findings illuminate the prevalence and dynamic nature of translational regulation underlying the mammalian cell cycle.

Context Although paclitaxel is one of the most commonly used drugs to treat breast, ovarian, and lung cancers, little is known about the impact of paclitaxel-induced peripheral neuropathy (PIPN) on cancer survivors. Objectives The purposes of this study were to evaluate for differences in demographic and clinical characteristics as well as measures of sensation, balance, upper extremity function, perceived stress, symptom burden, and quality of life (QOL) between survivors who received paclitaxel and did (n = 153) and did not (n = 58) develop PIPN. Methods Pain characteristics associated with PIPN are described in detail. Both subjective and objective measures were used to evaluate the impact of PIPN. Results Survivors with PIPN were significantly older, had a higher body mass index, and a worse comorbidity profile. The duration of PIPN was almost four years, and pain scores were in the moderate range. Compared with survivors without PIPN, survivors with PIPN had a higher number of upper and lower extremity sites that had lost light touch, cold, and pain sensations. Survivors with PIPN had worse upper extremity function, more problems with balance, a higher symptom burden, and higher levels of perceived stress. In addition, survivors with PIPN had worse QOL scores particularly in the domain of physical functioning. Conclusion The findings from this large descriptive study are the first to document the impact of PIPN on survivors' symptom burden, functional status, and QOL.

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm of early childhood characterized by Ras pathway mutations and aberrant DNA methylation. Despite allogeneic hematopoietic stem cell transplantation (HSCT) approx. 30% of patients (pts) succumb to resistant leukemia. Slowly regressing disease in the absence of HSCT is, however, noted in some pts. Mutational patterns and known clinical risk factors are insufficient to fully explain this heterogeneity. Three independent recent studies demonstrated the potential of DNA methylation profiling to identify clinically meaningful risk groups (Lipka et al., Nat Commun 2017; Stieglitz et al., Nat Commun 2017; Murakami et al., Blood 2018). The aim of this work was to establish an international consensus for the classification of JMML based on DNA methylation, to systematically describe the biological and clinical features of the subgroups identified and to develop a molecular classifier that enables the prospective assignment of DNA methylation categories. In a collaborative effort, we re-analyzed three well-annotated Illumina 450k methylation array data sets. In total, data from 292 pts with JMML or Noonan syndrome-associated myeloproliferative disorder (NS/MPD; EWOG-MDS, Europe: 147 pts, Nagoya, Japan: 106 pts, UCSF, USA: 39 pts) were available. Methylation data from 285 pts passed our quality control and were included in the present analysis. Of these, 256 (89.8%) pts had been diagnosed with JMML and 29 (10.2%) with NS/MPD. Mean age at diagnosis was 1.9 yrs. Levels of fetal hemoglobin (HbF) were available in 228/285 pts and elevated for age in 61%. Driver mutations affecting the Ras pathway were identified in 92% of JMML pts (PTPN11: 36%, NF1: 11%, NRAS: 15%, KRAS: 15%, CBL: 14%), and monosomy 7 in 14%. Initial exploratory analysis revealed batch effects that could, at least in part, be explained by the different ethnical backgrounds of the pts but also included a technical component. Therefore, we stringently filtered probes containing SNPs and performed batch correction using an empirical Bayes framework (Combat). CpG dinucleotides (CpGs) showing variable methylation across normal hematopoietic cell types were excluded from the analysis to avoid confounding with differential cell type composition between the samples. Unsupervised consensus clustering using the 5000 most variable CpGs identified three stable methylation clusters in this inter-group patient cohort. According to their DNA methylation levels, the clusters were designated "high methylation" (HM), "intermediate methylation" (IM) and "low methylation" (LM). Overall, 29.5%, 23.5%, and 47.0% of the cohort of JMML and NS/MPD pts were assigned to the HM, IM, and LM clusters, respectively. The methylation clusters showed a highly significant association with specific Ras-pathway mutations and with known clinical risk factors. All NS/MPD pts clustered with the LM subgroup and showed the lowest methylation levels. The HM cluster was enriched for somatic PTPN11 mutations, elevated HbF and older age, while the LM cluster was enriched for NRAS and CBL mutations, normal HbF and age assignments made in the present study to those from the original publications found only 3.9% (11/285) discordant cases. This encouraged us to establish a low-dimensional DNA methylation classifier for JMML that could be applied in a prospective setting. Therefore, we split the intergroup cohort randomly into a training (N=202) and a validation cohort (N=83) and trained an extreme gradient boosting machine (xgboost). When using as few as 10 CpGs, the resulting model predicted methylation clusters with 94% accuracy in the validation cohort. This inter-group meta-analysis is the single largest dataset of JMML patients to date and demonstrates the robustness of the JMML methylation signature across continents of origin and its power to identify biologically and clinically distinct JMML subgroups. Our ongoing efforts include developing a sequencing-based assay to predict DNA methylation clusters and to validate this assay using an independent cohort of 60 pts from the three participating research groups. This assay would allow for inclusion of DNA methylation as a biomarker into prospective clinical trials to assist in molecularly driven risk-stratification of JMML. Disclosures Niemeyer: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Upfront resistance to glucocorticoids (GCs) confers a poor prognosis for children with T-cell acute lymphoblastic leukemia (T-ALL). Using primary diagnostic samples from the Children's Oncology Group trial AALL1231, we previously demonstrated that one-third of patient T-ALL samples are intrinsically resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the T-ALL microenvironment. Furthermore, we demonstrated that inhibiting JAK/STAT signaling downstream of the IL7 receptor (IL7R) with the JAK1/2 inhibitor ruxolitinib (RUX) overcomes GC resistance in these samples. The objective of the present study was to determine the mechanism of IL7-induced GC resistance in T-ALL and to identify novel therapeutic targets to enhance GC sensitivity. We utilized CCRF-CEM cells, a human T-ALL cell line, as a model system in conjunction with primary patient samples. Exposing CCRF-CEM cells to IL7 induced phosphorylation of STAT5, the predominant downstream effector of IL7R signaling. When cultured in the presence of IL7 and the GC dexamethasone (DEX), CCRF-CEM cells recapitulated the IL7-induced GC resistance phenotype observed in patient samples. This resistance could be overcome with RUX, and Bliss index analysis demonstrated a synergistic relationship between DEX and RUX in the presence of IL7. Furthermore, CRISPR/Cas9 mediated knockout of STAT5 (STAT5 KO) was sufficient to overcome resistance, implicating STAT5 as the critical mediator of IL7-induced GC resistance. DEX exposure potently induced upregulation of IL7R expression in CCRF-CEM cells. Using a luciferase reporter construct containing a series of STAT5 response elements, we demonstrated that in the presence of IL7, DEX-induced upregulation of IL7R expression is associated with increased downstream signal transduction, leading to a significant increase in STAT5 transcriptional output. We then performed RNA-seq to further assess the functional consequences of this enhanced STAT5-mediated transcription. Gene set enrichment analysis (GSEA) revealed that STAT5 target genes were significantly upregulated in cells exposed to DEX and IL7 relative to IL7 alone (normalized enrichment score -2.27; p < 0.001; FDR < 0.001), suggesting that DEX exposure augments activation of the STAT5 transcriptional program. One critical component of this program that was induced by the combination of DEX and IL7 was the anti-apoptotic family member BCL2, which was not induced by DEX alone. Further analysis of its protein expression in CCRF-CEM cells confirmed this paradoxical upregulation of BCL2 specifically by the combination of DEX and IL7. Furthermore, BCL2 was not upregulated by DEX and IL7 in STAT5 KO cells, consistent with this being a STAT5-mediated effect. IL7-induced GC resistance could be overcome with shRNA-mediated knockdown of BCL2 and with pharmacologic inhibition of BCL2 by venetoclax. Similar to the effect observed with RUX, Bliss index analysis demonstrated synergy between DEX and venetoclax in the presence of IL7. Consistent with our observations in CCRF-CEM cells, an analysis of primary diagnostic T-ALL samples revealed DEX-induced upregulation of IL7R expression in samples with IL7-induced GC resistance, which was associated with increased BCL2 expression in the presence of DEX and IL7. Finally, we performed a similar analysis in healthy murine thymocytes, and found that CD4/CD8 double negative (DN) and CD4 or CD8 single positive (SP) thymocytes, but not double positive (DP) thymocytes, exhibited profound IL7-induced GC resistance that was associated with DEX-induced upregulation of IL7R expression and increased BCL2 expression in the presence of DEX and IL7. These data are consistent with the known role of IL7 specifically at the DN and SP stages of development, and suggests that IL7-induced GC resistance is a physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALL samples. Taken together, these data demonstrate that GCs paradoxically induce their own resistance in a subset of T-ALLs and in normal developing T-cells by augmenting a STAT5-mediated pro-survival program that results in upregulation of BCL2. Furthermore, we demonstrate that inhibition of JAK/STAT signaling or of BCL2 may have considerable therapeutic benefit to enhance GC sensitivity in T-ALL patients with IL7-induced GC resistance. Disclosures Teachey: La Roche: Consultancy; Amgen: Consultancy.

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1-TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1-TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1-TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1-TFE3, identified 2193 genes bound by ASPSCR1-TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1-TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1-TFE3, including MET (a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1-TFE3. As validation of this approach to identify genuine ASPSCR1-TFE3 target genes, two up-regulated genes bound by ASPSCR1-TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1-TFE3. As the results indicated that ASPSCR1-TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1-TFE3 target genes contribute to the growth of ASPSCR1-TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors.

Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

While patients with primary glioblastoma (GBM) have benefited from increased overall survival due to treatment with the alkylating chemotherapy temozolomide (TMZ), the benefit of TMZ for patients with diffuse low-grade gliomas (LGG) remains unknown. We previously demonstrated that among ten TMZ-treated patients with initial LGG (IDH1 mutant, 1p19q intact), treatment induced hypermutation in the recurrent tumors of six patients, all six of whom underwent malignant progression to secondary GBM (sGBM) (Johnson & Mazor, Science, 2014). To further explore the relationships among TMZ treatment, hypermutation and malignant progression, we studied tumor evolution in an expanded cohort of 35 TMZ-treated patients by exome-sequencing of paired IDH1/2-mutant LGGs and their post-TMZ recurrences. This expanded cohort allowed us to contrast patterns of hypermutation between 1p19q intact versus co-deleted subgroups. Hypermutated recurrences were common in both subgroups and consistently underwent malignant progression to the highest grade while acquiring somatic mutations in mismatch repair (MMR) genes. In contrast to the 1p19q intact subgroup in which TP53 mutations were present at diagnosis, the hypermutated recurrences of the 1p19q co-deleted subgroup frequently acquired TMZ-associated mutations in TP53. This suggests that TP53 mutations may cooperate with MMR mutations to convert TMZ-induced cytotoxicity to mutagenicity. Once hypermutated clones arose, we found that they dominated subsequent recurrences. In one case, a hypermutated sGBM was resected, but soon recurred with leptomeningeal spread, at which point hypermutated clones were found in the spinal cord. We examined the clonality of hypermutated tumors through exome-sequencing of multiple spatially distinct samples, from which we estimate that hypermutated recurrent tumors can originate from a single initiating cell. Together, these findings further our understanding of the molecular features and clinical behavior of hypermutated clones, raising concerns about the potent mutagenic activity of TMZ and other alkylating agents in LGG patients.

Background: A major challenge in interpreting high-throughput multianalyte genomic data sets such as those produced by the ISPY clinical trials is data integration and interpretation within the context of biologically relevant pathways. To address this need, the data analysis tool PARADIGM (PAthway Recognition Algorithm using Data Integration on Genomic Models) was developed to infer the activities of genetic pathways by integrating any number of functional genomic data sets for a given patient sample into a pathway activity profile. Methods: We used PARADIGM to integrate gene expression (Agilent 44K) and DNA copy number data (AFFY 22K and 330K MIP) from 133 ISPY-1 patients into pathway component activity levels for approximately 1400 curated signal transduction, transcriptional and metabolic pathways superimposed onto a single non-redundant ‘SuperPathway'. These pathway activities then become the substrate for statistical analyses to identify pathways characterizing different breast cancer subtypes, as well as those associated with recurrence and response to neoadjuvant chemotherapy within breast cancer subgroups. To identify subtype-specific pathway activities, we used ANOVA for initial feature filtering followed by Tukey analysis with Benjamini Hochberg multiple testing correction. For other binary outcome comparisons we used Mann-Whitney (2-sample Wilcoxon) analysis. PARADIGM results were corroborated with pathway enrichment analysis and filtered for significance. Results: In agreement with breast cancer cell line and other prior studies, basal-like and triple negative cancers are dominated by upregulation of the FOXM1 and MYC/Max subnetworks and downregulation of the FOXA1/ER signal transduction pathway, the converse of the activity pattern seen in luminal breast cancers. These and other subtype associations pass stringent multiple testing corrected significance tests. Though an association study of recurrence over the entire patient cohort mostly yields pathways characteristic of basal-like tumors, alternative pathway associations emerge when subtypes are analyzed individually for outcome and significance tests are relaxed to include features that pass un-corrected Wilcoxon significance tests and also generate highly significant pathway enrichment scores. Subtype-specific drivers of recurrence and chemo-resistance supported by this level of evidence include ALK1/2 (TGFB-BMP) and p53 effector signaling for basals and Syndecan-1 and c-MYC for luminals. Chemo-sensitivity pathways, assessed by association with pCR and RCB1, appear to be subtype-specific as well, with HDAC class 1 signaling, LRP6-Wnt, and IRE1alpha chaperones dominating basal-like cancers and c-MYB activity dominating Her2+ cancers, whereas chemo-sensitivity of HR+Her2- cancers though rare appears to be driven by the DNA damage axis (BRCA/BARD1). Conclusion: These and other similar analyses suggest that patients with TN or basal-like disease might benefit from the addition of ALK1 pathway inhibitors to treatment, whereas high risk HR+ patients might benefit from Syndecan-1 inhibitors. C-MYC/MAX inhibitors might benefit all high risk patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-09.

Malignant pleural mesotheliomas (MPMs) often show CDKN2A and NF2 inactivation, but other highly recurrent mutations have not been described. To identify additional driver genes, we used an integrated genomic analysis of 53 MPM tumor samples to guide a focused sequencing effort that uncovered somatic inactivating mutations in BAP1 in 23% of MPMs. The BAP1 nuclear deubiquitinase is known to target histones (together with ASXL1 as a Polycomb repressor subunit) and the HCF1 transcriptional co-factor, and we show that BAP1 knockdown in MPM cell lines affects E2F and Polycomb target genes. These findings implicate transcriptional deregulation in the pathogenesis of MPM.

Introduction To identify new therapeutic approaches in malignant mesothelioma (MM), we examined the expression and activation of receptor tyrosine kinases (RTKs) and the effects of specific RTK inhibitors and the mammalian target of rapamycin (mTOR) inhibitor rapamycin; the latter being of special interest in MM given the recent linkage between NF2 loss and mTOR activation. Methods We performed a screen for mutated or activated RTKs in 14 MM cell lines and 70 primary tumors. Expression of phosphorylated RTKs was analyzed by Western blotting and a membrane-based antibody array in normal growth conditions and after treatment by specific inhibitors. MET and epidermal growth factor receptor (EGFR) mutations were screened by sequencing. MET, hepatocyte growth factor, insulin-like growth factor 1 receptor, and EGFR expression were studied by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay, and by Affymetrix expression microarrays. Results Profiling of the phosphorylation status of 42 RTKs showed prominent coactivation of MET and EGFR in 8 of 14 (57%) MM cell lines. MET, EGFR, and insulin-like growth factor 1 receptor were the main RTKs activated after mTOR inhibition and contributed to AKT feedback activation. Knockdown of MET by RNA interference inhibited not only the phosphorylation of MET but also that of EGFR. Conversely, stimulation with hepatocyte growth factor increased both phospho-MET and phospho-EGFR. The combination of PHA-665752 and the EGFR inhibitor, erlotinib, suppressed cell growth more than either agent alone in three of six cell lines tested. Finally, combinations of rapamycin and different RTK inhibitors were more active than either drug alone in 12 of 13 cell lines. Conclusion Combination targeting of kinase signaling pathways is more effective than single agents in most MM.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Overall survival is typically 6 months from diagnosis(1). Numerous phase 3 trials of agents effective in other malignancies have failed to benefit unselected PDA populations, although patients do occasionally respond. Studies in other solid tumors have shown that heterogeneity in response is determined, in part, by molecular differences between tumors. Furthermore, treatment outcomes are improved by targeting drugs to tumor subtypes in which they are selectively effective, with breast(2) and lung(3) cancers providing recent examples. Identification of PDA molecular subtypes has been frustrated by a paucity of tumor specimens available for study. We have overcome this problem by combined analysis of transcriptional profiles of primary PDA samples from several studies, along with human and mouse PDA cell lines. We define three PDA subtypes: classical, quasimesenchymal and exocrine-like, and we present evidence for clinical outcome and therapeutic response differences between them. We further define gene signatures for these subtypes that may have utility in stratifying patients for treatment and present preclinical model systems that may be used to identify new subtype specific therapies.

Recent prostate-specific antigen–based screening trials indicate an urgent need for novel and noninvasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Noncoding microRNAs (miRNA) in the serum and plasma have been shown to have potential as noninvasive markers for physiologic and pathologic conditions. To identify serum miRNAs that diagnose and correlate with the prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR method involving the purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA-deficient) mouse ES cells as the benchmark, we confirmed the validity of our technique and uncovered a considerable lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA scores, we identified miRNA signatures that allow us to diagnose cancer patients and correlate with a prognosis. These serum signatures include oncogenic and tumor-suppressive miRNAs, suggesting functional roles in prostate cancer progression. Cancer Res; 71(2); 550–60. ©2010 AACR.

(Objective) The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. (Materials and methods) Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109.(Results) UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays and mouse xenografts supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers.(Conclusion) We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers. Citation Format: Steven J. Wang, Saurabh Asthana, Annemieke van Zante, Adam B. Olshen, Frank McCormick, Osamu Tetsu. Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3104.

Background Most patients with juvenile myelomonocytic leukemia (JMML) are curable only with allogeneic hematopoietic cell transplantation (HCT). However, the current standard conditioning regimen, busulfan-cyclophosphamide-melphalan (Bu-Cy-Mel), may be associated with higher risks of morbidity and mortality. ASCT1221 was designed to test whether the potentially less-toxic myeloablative conditioning regimen containing busulfan-fludarabine (Bu-Flu) would be associated with equivalent outcomes. Procedure Twenty-seven patients were enrolled on ASCT1221 from 2013 to 2015. Pre- and post-HCT (starting Day +30) mutant allele burden was measured in all and pre-HCT therapy was administered according to physician discretion. Results Fifteen patients were randomized (six to Bu-Cy-Mel and nine to Bu-Flu) after meeting diagnostic criteria for JMML. Pre-HCT low-dose chemotherapy did not appear to reduce pre-HCT disease burden. Two patients, however, received aggressive chemotherapy pre-HCT and achieved low disease-burden state; both are long-term survivors. All four patients with detectable mutant allele burden at Day +30 post-HCT eventually progressed compared to two of nine patients with unmeasurable allele burden (P = 0.04). The 18-month event-free survival of the entire cohort was 47% (95% CI, 21-69%), and was 83% (95% CI, 27-97%) and 22% (95% CI, 03-51%) for Bu-Cy-Mel and Bu-Flu, respectively (P = 0.04). ASCT1221 was terminated early due to concerns that the Bu-Flu arm had inferior outcomes. Conclusions The regimen of Bu-Flu is inadequate to provide disease control in patients with JMML who present to HCT with large burdens of disease. Advances in molecular testing may allow better characterization of biologic risk, pre-HCT responses to chemotherapy, and post-HCT management.

When a cancer patient develops a new tumor it is necessary to determine if it is a recurrence (metastasis) of the original cancer, or an entirely new occurrence of the disease. This is accomplished by assessing the histo-pathology of the lesions. However, there are many clinical scenarios in which this pathological diagnosis is difficult. Since each tumor is characterized by a distinct pattern of somatic mutations, a more definitive diagnosis is possible in principle in these difficult clinical scenarios by comparing the two patterns. In this article we develop and evaluate a statistical strategy for this comparison when the data are derived from array copy number data, designed to identify all of the somatic allelic gains and losses across the genome. First a segmentation algorithm is used to estimate the regions of allelic gain and loss. The correlation in these patterns between the two tumors is assessed, and this is complemented with more precise quantitative comparisons of each plausibly clonal mutation within individual chromosome arms. The results are combined to determine a likelihood ratio to distinguish clonal tumor pairs (metastases) from independent second primaries. Our data analyses show that in many cases a strong clonal signal emerges. Sensitivity analyses show that most of the diagnoses are robust when the data are of high quality.

Background Paclitaxel is one of the most commonly used drugs to treat breast cancer. Its major dose-limiting toxicity is paclitaxel-induced peripheral neuropathy (PIPN). PIPN persists into survivorship and has a negative impact on patient’s mood, functional status, and quality of life. No interventions are available to treat PIPN. A critical barrier to the development of efficacious interventions is the lack of understanding of the mechanisms that underlie PIPN. Mitochondrial dysfunction has been evaluated in preclinical studies as a hypothesized mechanism for PIPN, but clinical data to support this hypothesis are limited. The purpose of this pilot study was to evaluate for differential gene expression and perturbed pathways between breast cancer survivors with and without PIPN. Methods Gene expression in peripheral blood was assayed using RNA-seq. Differentially expressed genes (DEG) and pathways associated with mitochondrial dysfunction were identified between survivors who received paclitaxel and did (n = 25) and did not (n = 25) develop PIPN. Results Breast cancer survivors with PIPN were significantly older; more likely to be unemployed; reported lower alcohol use; had a higher body mass index and poorer functional status; and had a higher number of lower extremity sites with loss of light touch, cold, and pain sensations and higher vibration thresholds. No between-group differences were found in the cumulative dose of paclitaxel received or in the percentage of patients who had a dose reduction or delay due to PIPN. Five DEGs and nine perturbed pathways were associated with mitochondrial dysfunction related to oxidative stress, iron homeostasis, mitochondrial fission, apoptosis, and autophagy. Conclusions This study is the first to provide molecular evidence that a number of mitochondrial dysfunction mechanisms identified in preclinical models of various types of neuropathic pain including chemotherapy-induced peripheral neuropathy are found in breast cancer survivors with persistent PIPN and suggest genes for validation and as potential therapeutic targets.

Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas ( TCGA) pilot project aims to assess the value of large- scale multi- dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas - the most common type of primary adult brain cancer - and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol- 3- OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.

Despite advances in therapy, many patients with systemic light-chain amyloidosis (AL) die within 3 years from diagnosis. The humanized 2B6 monoclonal antibody (MoAb) is specific for the low-affinity IgG Fc receptor CD32B and effective in a human CD32B+ B-cell lymphoma murine xenograft model. Because MoAb therapy could improve outcomes in AL, we studied CD32B expression by clonal plasma cells obtained from 48 patients with AL. Transcript profiling showed that expression of CD32B was significantly higher than expression of all other Fc receptor family members. Reverse-transcriptase polymerase chain reaction (RT-PCR) using double-enriched CD138+ plasma cells showed uniform expression of the stable cell surface CD32B1 isoform at diagnosis and relapse, and flow cytometry showed intense CD32B cell surface staining on 99% of CD138+ plasma cells at diagnosis and relapse. These data provide a rationale for the novel therapeutic targeting of CD32B using the humanized 2B6 MoAb in patients with systemic AL-amyloidosis.

We performed a three-phase genome-wide association study (GWAS) using cases and controls from a genetically isolated population, Ashkenazi Jews (AJ), to identify loci associated with breast cancer risk. In the first phase, we compared allele frequencies of 150,080 SNPs in 249 high-risk, BRCA1/2 mutation-negative AJ familial cases and 299 cancer-free AJ controls using χ 2 and the Cochran–Armitage trend tests. In the second phase, we genotyped 343 SNPs from 123 regions most significantly associated from stage 1, including 4 SNPs from the FGFR2 region, in 950 consecutive AJ breast cancer cases and 979 age-matched AJ controls. We replicated major associations in a third independent set of 243 AJ cases and 187 controls. We obtained a significant allele P value of association with AJ breast cancer in the FGFR2 region ( P = 1.5 × 10 −5 , odds ratio (OR) 1.26, 95% confidence interval (CI) 1.13–1.40 at rs1078806 for all phases combined). In addition, we found a risk locus in a region of chromosome 6q22.33 ( P = 2.9 × 10 −8 , OR 1.41, 95% CI 1.25–1.59 at rs2180341). Using several SNPs at each implicated locus, we were able to verify associations and impute haplotypes. The major haplotype at the 6q22.33 locus conferred protection from disease, whereas the minor haplotype conferred risk. Candidate genes in the 6q22.33 region include ECHDC1 , which encodes a protein involved in mitochondrial fatty acid oxidation, and also RNF146 , which encodes a ubiquitin protein ligase, both known pathways in breast cancer pathogenesis.

The principal curative agent in the front-line treatment of patients with diffuse large B-cell lymphoma (DLBCL) is the anthracycline, doxorubicin. To define pathways that may have a functional role in the response of DLBCL in vivo to doxorubicin-based therapies, seven DLBCL cell lines were treated with doxorubicin and the cellular response evaluated. Expression profiling of responses revealed changes in levels of genes consistent with discrete pathway activation that were confirmed functionally. The two most sensitive cell lines (Ly3 and Ly10) displayed activation of the TP53 pathway but not in the remaining five (Ly1, Ly2, Ly4, Ly7 and Ly8), where TP53 mutations were identified. In this latter group, a G2/M delay was invoked. NF-kappaB pathway activation was evident in Ly1 which with Ly4 displayed the most chemoresistant response. Treatment of Ly1 after doxorubicin with the proteasomic inhibitor, bortezomib, additively increased the cytotoxic effect of doxorubicin. Chemoresistance of Ly4 was associated with loss of chromosome 2 (0-9 Mbp) that in vivo was highly correlated with adverse outcome. Thus, the response of DLBCL in vivo and in vitro is defined by several distinct molecular and genetic pathways which is, perhaps, not surprising given the heterogeneous clinical, morphologic and genetic nature of DLBCL.