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Chromosomal microarray (CMA) is a testing modality frequently used in pediatric patients; however, published data on its utilization are limited to the genetic setting. We performed a database search for all CMA testing performed from 2010 to 2020, and delineated the diagnostic yield based on patient characteristics, including sex, age, clinical specialty of providers, indication of testing, and pathogenic finding. The indications for testing were further categorized into Human Phenotype Ontology categories for analysis. This study included a cohort of 14,541 patients from 29 different medical specialties, of whom 30% were from the genetics clinic. The clinical indications for testing suggested that neonatology patients demonstrated the greatest involvement of multiorgan systems, involving the most Human Phenotype Ontology categories, compared with developmental behavioral pediatrics and neurology patients being the least. The top pathogenic findings for each specialty differed, likely due to the varying clinical features and indications for testing. Deletions involving the 22q11.21 locus were the top pathogenic findings for patients presenting to genetics, neonatology, cardiology, and surgery. Our data represent the largest pediatric cohort published to date. This study is the first to demonstrate the diagnostic utility of this assay for patients seen in the setting of different specialties, and it provides normative data of CMA results among a general pediatric population referred for testing because of variable clinical presentations.

To investigate germline predisposition in lymphoma, we performed whole-exome sequencing and discovered a novel variant (c.817-1GT) in programmed cell death 1 ligand 2 (PD-L2) in a family with early-onset lymphomas and other cancers. The variant was present in the proband with follicular lymphoma and his son with Hodgkin's lymphoma. It was in the terminal splice acceptor site of PD-L2 and embedded in a putative enhancer of Janus kinase 2 (JAK2) and programmed cell death 1 ligand (PD-L1). We also found that gene expression of PD-L2, PD-L1, and JAK2 was significantly increased. Using 3' rapid amplification of cDNA ends (3' RACE), we detected an abnormal PD-L2 transcript in the son. Thus, the c.817-1GT variant may result in the elevated PD-L2 expression due to the abnormal PD-L2 transcript and the elevated PD-L1 and JAK2 expression due to increased enhancer activity of PD-L1 and JAK2. The PD-L2 novel variant likely underlies the genetic etiology of the lymphomas in the family. As PD-L2 plays critical roles in tumor immunity, identification of PD-L2 as a germline predisposition gene may inform personalized immunotherapy in lymphoma patients.

Exome reanalysis is useful for providing molecular diagnoses for previously uninformative samples. However, challenges exist in implementing a practical solution for clinicians and laboratories. This study complements the current literature by providing practical considerations for patient-level and cohort-level reanalyses. The Clinical and Laboratory Standards Institute assembled the Document Development Committee and an interpretation working group that developed the framework for reevaluation of exome-based data. We describe two distinct but complementary approaches toward exome reanalyses: clinician-initiated patient-level reanalysis, and laboratory-initiated cohort-level reanalysis. We highlight the advantages and constraints for both approaches, and provide a high-level conceptual guide for ordering clinicians and laboratories through the critical decision pathways. Because clinical exome sequencing continues to be the standard of care in genetics, exome reanalysis would be critical in increasing the overall diagnostic yield. A systematic guide will facilitate the efficient adoption of reevaluation of exome data for laboratories, health care professionals, genetic counselors, and clinicians.

Aicardi-Goutières syndrome (AGS) is a progressive multisystem disorder including encephalopathy with significant impacts on intellectual and physical abilities. An early diagnosis is becoming ever more crucial, as targeted therapies are emerging. A deep understanding of the molecular heterogeneity of AGS can help guide the early diagnosis and clinical management of patients, and inform recurrence risks. Here, we detail the diagnostic odyssey of a patient with an early presentation of AGS. Exome and genome sequencing detected an intronic RNASEH2B variant missed in a conventional leukodystrophy NGS gene panel. RNA studies demonstrated that a c.322-17 A > G variant affected splicing and caused 16-nucleotide intronic retention in the RNASEH2B transcript, introducing an out-of-frame early termination codon. RNASEH2B expression in the patient's blood was reduced when compared to controls. Our study highlights the pathogenicity of this intronic variant and the importance of its inclusion in variant assessment.

Noninvasive prenatal screening (NIPS) using cell-free DNA has been assimilated into prenatal care. Prior studies examined clinical validity and technical performance in high-risk populations. This systematic evidence review evaluates NIPS performance in a general-risk population.Medline (PubMed) and Embase were used to identify studies examining detection of Down syndrome (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidies, rare autosomal trisomies, copy number variants, and maternal conditions, as well as studies assessing the psychological impact of NIPS and the rate of subsequent diagnostic testing. Random-effects meta-analyses were used to calculate pooled estimates of NIPS performance (P.05). Heterogeneity was investigated through subgroup analyses. Risk of bias was assessed.A total of 87 studies met inclusion criteria. Diagnostic odds ratios were significant (P.0001) for T21, T18, and T13 for singleton and twin pregnancies. NIPS was accurate (≥99.78%) in detecting sex chromosome aneuploidies. Performance for rare autosomal trisomies and copy number variants was variable. Use of NIPS reduced diagnostic tests by 31% to 79%. Conclusions regarding psychosocial outcomes could not be drawn owing to lack of data. Identification of maternal conditions was rare.NIPS is a highly accurate screening method for T21, T18, and T13 in both singleton and twin pregnancies.

Novel gene-disease discoveries, rapid advancements in technology, and improved bioinformatics tools all have the potential to yield additional molecular diagnoses through the reanalysis of exome sequencing data. Collaborations between clinical laboratories, ordering physicians, and researchers are also driving factors that can contribute to these new insights. Automation in ongoing natural history collection, evolving phenotype updates, advancements in processing next-generation sequencing data, and up-to-date variant-gene-disease databases are increasingly needed for systematic exome reanalysis. Here, we review some of the advantages and challenges for clinician-initiated and laboratory-initiated exome reanalysis, and we propose a model for the future that could potentially maximize the clinical utility of exome reanalysis by integrating information from electronic medical records and knowledge databases into routine clinical workflows.

The extent and importance of functional heterogeneity and crosstalk between tumor cells is poorly understood. Here, we describe the generation of clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations are engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grows robustly, generating exclusively malignant ascites. However, multiclonal mixtures form large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions are sufficient to promote metastasis of CL31. CL31 uniquely harbors ERBB2 amplification, and its acquired metastatic activity in clonal mixtures is dependent on transient exposure to amphiregulin, which is exclusively secreted by non-tumorigenic clones. Amphiregulin enhances CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via “hit-and-run” commensal interactions., Cooperative interactions among tumor cells may have important implications for metastasis. Here, the authors examined the spatio-temporal nature of interactions among clonal populations of ovarian carcinoma cells and found that transient interactions cells can promote metastases via commensal interactions.

PurposeThis study aimed to establish variants inCBX1, encoding heterochromatin protein 1β (HP1β), as a cause of a novel syndromic neurodevelopmental disorder.MethodsPatients withCBX1variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performedin vitrocellular assays, neurobehavioral and cytological analyses of neuronal cells obtained from newly generatedCbx1mutant mouse lines.ResultsIn three unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygousde novovariants inCBX1. The identified variants were in the chromodomain, the functional domain of HP1 β, which mediates interactions with chromatin.Cbx1chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1β binding to heterochromatin, while HP1β interactome analysis demonstrated that the majority of HP1β-interacting proteins remained unchanged between the wild-type and mutant HP1β.ConclusionThese collective findings confirm the role ofCBX1in developmental disabilities through the disruption of HP1β chromatin binding during neurocognitive development. As HP1β forms homodimers and heterodimers, mutant HP1β likely sequesters wild-type HP1β and other HP1 proteins, exerting dominant-negative effects.

Carrier screening involves detection of carrier status for genes associated with recessive conditions. A negative carrier screening test result bears a nonzero residual risk (RR) for the individual to have an affected child. The RR depends on the prevalence of specific conditions and the detection rate (DR) of the test itself. Herein, we provide a detailed approach for calculating DR and RR. DR was calculated on the basis of the sum of disease allele frequencies (DAFs) of pathogenic variants found in published literature. As a proof of concept, DAF data for cystic fibrosis were compared with society guidelines. The DAF data calculated by this method were consistent with the published cystic fibrosis guideline. In addition, we compared DAF for four genes (ABCC8, ASPA, GAA, and MMUT) across three laboratories, and outlined the likely reasons for discrepancies between these laboratories. The utility of carrier screening is to support couples with information while making reproductive choices. Accurate development of DR and RR is therefore critical. The method described herein provides an unbiased and transparent process to collect, calculate, and report these data.

To interrogate functional heterogeneity and crosstalk between tumor cells, we generated clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations were engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grew robustly, generating exclusively malignant ascites. However, multiclonal mixtures formed large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions were sufficient to promote metastasis of CL31. CL31 uniquely harbored ERBB2 amplification, and its acquired metastatic trait was dependent on transient exposure to amphiregulin, which was exclusively secreted by non-tumorigenic clones. Amphiregulin enhanced CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via “hit- and-run” commensal interactions.

Cystic fibrosis (CF) is one of the most common genetic diseases worldwide with high carrier frequencies across different ethnicities. Next generation sequencing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has proven to be an effective screening tool to determine carrier status with high detection rates. Here, we evaluate the performance of the Swift Biosciences Accel-Amplicon CFTR Capture Panel using CFTR-positive DNA samples. This assay is a one-day protocol that allows for one-tube reaction of 87 amplicons that span all coding regions, 5′ and 3′UTR, as well as four intronic regions. In this study, we provide the FASTQ, BAM, and VCF files on seven unique CFTR-positive samples and one normal control sample (14 samples processed including repeated samples). This method generated sequencing data with high coverage and near 100% on-target reads. We found that coverage depth was correlated with the GC content of each exon. This dataset is instrumental for clinical laboratories that are evaluating this technology as part of their carrier screening program., Measurement(s)amplicon sequencingTechnology Type(s)DNA sequencingFactor Type(s)CFTR mutationsSample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10060244

Metastasis is a complex biological process that has been difficult to delineate in human colorectal cancer (CRC) patients. A major obstacle in understanding metastatic lineages is the extensive intra-tumor heterogeneity at the primary and metastatic tumor sites. To address this problem, we developed a highly multiplexed single-cell DNA sequencing approach to trace the metastatic lineages of two CRC patients with matched liver metastases. Single-cell copy number or mutational profiling was performed, in addition to bulk exome and targeted deep-sequencing. In the first patient, we observed monoclonal seeding, in which a single clone evolved a large number of mutations prior to migrating to the liver to establish the metastatic tumor. In the second patient, we observed polyclonal seeding, in which two independent clones seeded the metastatic liver tumor after having diverged at different time points from the primary tumor lineage. The single-cell data also revealed an unexpected independent tumor lineage that did not metastasize, and early progenitor clones with the “first hit” mutation in APC that subsequently gave rise to both the primary and metastatic tumors. Collectively, these data reveal a late-dissemination model of metastasis in two CRC patients and provide an unprecedented view of metastasis at single-cell genomic resolution.

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor’s major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48–96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5–6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

Aberrant expression of microRNAs (miRNAs) and the enzymes that control their processing have been reported in multiple biological processes including primary and metastatic tumours1–6, but the mechanisms governing this are not clearly understood. Here we show that TAp63, a p53 family member, suppresses tumorigenesis and metastasis, and coordinately regulates Dicer and miR-130b to suppress metastasis. Metastatic mouse and human tumours deficient in TAp63 express Dicer at very low levels, and we found that modulation of expression of Dicer and miR-130b markedly affected the metastatic potential of cells lacking TAp63. TAp63 binds to and transactivates the Dicer promoter, demonstrating direct transcriptional regulation of Dicer by TAp63. These data provide a novel understanding of the roles of TAp63 in tumour and metastasis suppression through the coordinate transcriptional regulation of Dicer and miR-130b and may have implications for the many processes regulated by miRNAs.

The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.

Vemurafenib and dabrafenib selectively inhibit the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) kinase, resulting in high response rates and increased survival in melanoma. Approximately 22% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) during therapy. The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation. Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK. JNK signaling is suppressed in multiple contexts, including in cSCC of vemurafenib-treated patients, as well as in mice. Expression of a mutant ZAK that cannot be inhibited reverses the suppression of JNK activation and apoptosis. Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies. DOI: http://dx.doi.org/10.7554/eLife.00969.001, eLife digest Over 50% of melanomas, a highly lethal form of skin cancer, carry mutations in a gene called BRAF. The BRAF gene encodes an enzyme that helps to regulate the proliferation of cells, but mutations in this gene lead to the excessive proliferation that is seen in cancer. Clinical trials have shown that a drug called vemurafenib can be used to treat patients who carry the mutated BRAF genes and go on to develop melanoma, but around one fifth of these patients developed another type of skin cancer called cSCC (cutaneous squamous cell carcinoma). The cSCC tumors often develop in areas where the sun has damaged the patient’s skin, and it is thought that their growth is then accelerated by vemurafenib activating another enzyme, ERK, which causes the excessive proliferation of skin cells. Vin et al. have now found that vemurafenib might also cause cSCC tumors by blocking another signaling pathway. The experiments were performed in human cells and also in mice, and the results were then verified in human cSCC samples. Cells that are exposed to UV radiation usually die, but when treated with vemurafenib, some 70% of the cells that would have died instead survived. The stress from the UV radiation activates the JNK signaling pathway, which causes the irradiated cells to die. However, Vin et al. found that cSCC cells had very low levels of JNK signaling because treatment with vemurafenib had the unintended effect of inhibiting three enzymes that are needed to fully activate the JNK signaling pathway. Vin et al. estimate that suppression of JNK signaling and cell death is responsible for about 17.6 to 40% of the effect on cSCC growth seen in melanoma patients, with activation of the ERK pathway accounting for the rest. These unexpected findings suggest that combining vemurafenib treatment with radiation or chemotherapy should be done with caution as these effects could affect their efficacy. It also suggests that future drugs should be designed in a way that avoids these types of effects by making sure they do not inhibit important ‘off-target’ enzymes. DOI: http://dx.doi.org/10.7554/eLife.00969.002

Metastatic colorectal cancer (mCRC) is a devastating disease with only 11% 5-year survival rate for stage IV patients. The genomic basis of metastasis has been difficult to study, in part due to the extensive intratumor heterogeneity at both the primary and metastatic tumor sites. Previous studies have applied bulk next-generation sequencing (NGS) methods, which have limited ability to resolve intratumor heterogeneity. To address this problem, we developed a highly-multiplexed single cell DNA sequencing method that combines flow-sorting of single nuclei, multiple-displacement-amplification, low-input library preparation, library barcoding, targeted capture and NGS to generate high-coverage data from single tumor cells. To increase throughput, we performed targeted single-cell DNA sequencing using a panel of 201-cancer genes to analyze single cells from primary tumors and liver metastases from two mCRC patients. In each patient, we sequenced 90 single tumor cells from the primary and metastatic tumor, to delineate the clonal architecture of the tumor and reconstruct their phylogenetic lineages. In addition, we sequenced populations of tumor cells at high coverage depth (200X). Our data identified a large number of nonsynonymous mutations that evolved in the root nodes during the earliest stages of primary tumor evolution and were maintained in all single cells during the clonal expansion of the tumor mass. We also identified a small number of mutations that were specific to the liver metastases, which are likely to play an important role in metastatic dissemination. Using the single cell data, we constructed phylogenetic trees, which revealed branched evolution at both organ sites. Collectively, our data suggest that both mCRC patients are consistent with a late-dissemination model, in which the primary tumors evolved for a long period of time prior to the dissemination of clones to distant organ sites. This model has important clinical implications, by suggesting that surgical intervention or therapeutic treatment of the primary CRC tumor can prevent metastasis, since single tumor cells do not disseminate at the earliest stages of tumor growth Citation Format: Marco L. Leung, Anna Casasent, Yong Wang, Emi Sei, Nicholas Navin. Single-cell DNA sequencing identifies a late dissemination model in metastatic colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 157.

Human breast cancers often display intratumor genomic heterogeneity. This clonal diversity confounds the clinical diagnosis and basic research of cancer, because single samples may not represent that tumor as a whole. Sequencing breast tumor cohorts en masse has identified many prevalent mutations, but has limited ability for resolving subclonal diversity. Here, we developed a whole-genome and exome single-cell sequencing approach (Nuc-Seq) using G2/M cells. To validate our method, we applied Nuc-Seq to sequence the whole genomes of two single cells from a genetically monoclonal breast cancer cell line (SK-BR-3) at high coverage depth (61X ± 5 sem, n=2) and breadth (83.70% ± 3.40 sem, n=2) to detect somatic mutations. Our analysis suggests that Nuc-Seq generates low allelic dropout rates (9.73% ± 2.19%) and low false positive error rates for point mutations (FPR = 1.24e-6). We then applied this method to sequence single normal and tumor cells from an estrogen-receptor positive breast cancer and a triple-negative ductal carcinoma at base-pair resolution. In parallel, we performed single cell copy number profiling. In both tumors, we observed a large number of rare variants that were not detected by sequencing the bulk tumor en masse. In contrast, we find that single cell copy number profiles are highly similar. Our data suggest that aneuploid rearrangements occurred early in tumor evolution and remained highly stable as the tumor mass expanded. In contrast we find that point mutations evolved gradually, generating extensive clonal diversity. Many of the diverse mutations were shown to occur at low frequencies (0.03 -10%) in the tumor mass by targeted duplex sequencing. Mathematical modeling suggests that the triple-negative tumor cells have an increased mutation rate (13.3X), while the ER+ tumor cells do not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer. Citation Format: Yong Wang, Nicholas Navin, Jill Waters, Marco Leung, Anna Unruh, Xiuqing Shi, Whijae Roh, Ken Chen, Paul Scheet, Selina Vattathil, Han Liang, Asha Multani, Hong Zhang, Funda Meric-Bernstam, Franziska Michor, Rui Zhao. Single cell genome sequencing reveals clonal stability and diversity in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-310. doi:10.1158/1538-7445.AM2014-LB-310

The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a critical transcriptional activator of a co-factor pivotal for miRNA processing, DGCR8. This regulation is important in the terminal differentiation of adult keratinocytes and likely of other cells. Loss of ΔNp63 or DGCR8 induces pluripotency in these cells. Generation of a conditional ΔNp63 knock-out mouse model with the help of a Cre-LoxP system led to the development of mice with single layered fragile and disorganized epidermis. Analysis of the skin with immunohistochemistry and immmunofluorescence exhibit aberrant expression of differentiation markers like keratin 5,14,10, loricrin and filaggrin, suggesting a defect in terminal differentiation and BrdU incorporation assay suggested hyperproliferation and self-renewal. Further characterization of stem cell marker expression in the epidermal cells from these embryos was found to be high. Chromatin immunoprecipitation assay and luciferase assays demonstrated the transcriptional activation of the DGCR8 promoter by ΔNp63. Overexpression of DGCR8 in the epidermal cells with the help of a lentiviral doxycycline inducible DGCR8 construct forced these cells to undergo differentiation as validated by in vitro differentiation assay and teratoma formation assay. These cells were also capable of generating chimeras when injected into blastocysts. Lentiviral knock-down of ΔNp63 or DGCR8 in normal human epidermal keratinocytes led to the development of induced pluripotent stem cells capable of forming teratomas as analyzed by differentiation markers AFP, MyoD and Nestin for various lineages like endoderm, mesoderm and ectoderm respectively. Low expression of key miRNAs in ΔNp63 deficient cells results in the failure to repress genes required for stem cell pluripotency, Oct4, Sox2, and Nanog. Strikingly, ΔNp63-/- epidermal cells display profound defects in terminal differentiation and express markers and miRNAs of pluripotency at levels comparable to those in embryonic stem cells. Moreover, these cells can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 become pluripotent. For the first time we demonstrate a novel role for ΔNp63 in the transcriptional regulation of DGCR8 to maintain stem cell pluripotency. Importantly, our findings identify a simple method of generating human induced pluripotent stem cells through the knockdown of a single gene. Citation Format: Deepavali Chakravarti, Xiaohua Su, Minsoon Cho, Young Jin Gi, Ashley Benham, Cristian Coarfa, Avinashnarayan Venkatanarayan, Jennifer Alana, Chad Creighton, weimin Xiao, Marco Leung, Harina Vin, Io Long Chan, Arianexys Aquino, Nicole Müller, Jan Parker-Thornburg, Kenneth Y. Tsai, Preethi H. Gunarante, Elsa R. Flores. Induced pluripotency in adult keratinocytes through downregulation of ΔNp63 or DGCR8. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1119. doi:10.1158/1538-7445.AM2013-1119

8537 Background: The advent of targeted therapy has revolutionized the treatment of cancer. The mutant BRAFV600E protein is found in over 50% of melanomas and thyroid carcinomas, resulting in elevated kinase activity, increased mitogen-activated protein kinase (MAPK) pathway signaling, and cell proliferation. Vemurafenib and PLX4720 were designed to selectively inhibit the BRAF kinase, and clinical trials of vemurafenib in metastatic melanoma have demonstrated a response rate of over 50% and an overall survival advantage over standard dacarbazine therapy. Approximately 20-30% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) highlighting the importance of understanding toxicities associated with this drug. Paradoxical ERK activation in BRAF wild-type, RAS-mutant cells is thought to be the major mechanism by which this occurs, as evidenced by the presence of RAS mutations in 60% of such lesions. Methods: Using a combination of BRAF-wild-type cSCC cell lines, primary human keratinocytes, as well as a UV mouse model of cSCC and human cSCC samples, we identified novel effects of BRAFi on apoptosis. Results: Here we show an unexpected and novel effect of vemurafenib and PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases. JNK signaling and apoptosis are suppressed in cSCC lesions arising in vemurafenib-treated patients as well as in irradiated mouse skin. This occurs independently of paradoxical ERK signaling and in the presence of MEK inhibitor. Treatment with PLX4720 greatly accelerates the development of UV-induced cSCC in mice without Ras mutations. Kinome screening identified ZAK and MKK4 (MEK4 / MAP2K4) kinases as inhibited by vemurafenib, leading to suppression of MKK4 and MKK7 (MAP2K7) phosphorylation. Knockdown of inhibited off-target kinases recapitulates these anti-apoptotic effects of vemurafenib. Conclusions: Our results implicate suppression of JNK signaling, independent of ERK activation, as an additional, complementary mechanism of adverse effects of vemurafenib. This has broad implications for combination therapies with other modalities that induce apoptosis and for the long-term use of vemurafenib in the adjuvant setting.

Single-cell genome sequencing methods are challenged by poor physical coverage and high error rates, making it difficult to distinguish real biological variants from technical artifacts. To address this problem, we developed a method called SNES that combines flow-sorting of single G1/0 or G2/M nuclei, time-limited multiple-displacement-amplification, exome capture, and next-generation sequencing to generate high coverage (96%) data from single human cells. We validated our method in a fibroblast cell line, and show low allelic dropout and false-positive error rates, resulting in high detection efficiencies for single nucleotide variants (92%) and indels (85%) in single cells. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0616-2) contains supplementary material, which is available to authorized users.