MOLECULAR AND CELLULAR MECHANISMS LEADING TO SIMILAR PHENOTYPES IN DOWN AND FETAL ALCOHOL SYNDROMES

poster abstractDown syndrome (DS) and Fetal Alcohol Syndrome (FAS) are two leading causes of birth defects with phenotypes ranging from cognitive impairment to craniofacial abnormalities. These syndromes have an estimated occurrence of 1/750 and 1/1000 live births, respectively. While DS originates from the trisomy of human chromosome 21 and FAS from excess alcohol consumption, many of the defining characteristics for these two disorders are stunningly similar. Our research of the published literature has identified more than 20 similarities in DS and FAS phenotypes including precise craniofacial and neurological abnormalities. We hypothesize that the similar phenotypes in these two syndromes are caused by disruptions in common molecular and cellular pathways. To test our hypothesis we are examining morphometric, genetic, and cellular phenotypes during development of DS and FAS mouse models. Our preliminary evidence indicates that during early development, expression of Dyrk1a and Rcan1 (two genes found in three copies in individuals with DS) is dysregulated in the craniofacial and neurological precursors of both DS and FAS as compared to normal control embryos. Using immuocytochemistry, we are analyzing cellular properties of neurological development in DS embryos and comparing deficiencies found between trisomic and normal mice to those found in FAS embryos at similar stages. These results will further define molecular and cellular alterations leading to DS and FAS phenotypes and provide mechanisms to target for potential pharmacotherapy

Next-generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy

Next-generation sequencing to detect circulating tumor DNA is a minimally invasive method for tumor genotyping and monitoring therapeutic response. The majority of studies have focused on detecting circulating tumor DNA from patients with metastatic disease. Herein, we tested whether circulating tumor DNA could be used as a biomarker to predict relapse in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. In this study, we analyzed samples from 38 early-stage triple-negative breast cancer patients with matched tumor, blood, and plasma. Extracted DNA underwent library preparation and amplification using the Oncomine Research Panel consisting of 134 cancer genes, followed by high-coverage sequencing and bioinformatics. We detected high-quality somatic mutations from primary tumors in 33 of 38 patients. TP53 mutations were the most prevalent (82%) followed by PIK3CA (16%). Of the 33 patients who had a mutation identified in their primary tumor, we were able to detect circulating tumor DNA mutations in the plasma of four patients (three TP53 mutations, one AKT1 mutation, one CDKN2A mutation). All four patients had recurrence of their disease (100% specificity), but sensitivity was limited to detecting only 4 of 13 patients who clinically relapsed (31% sensitivity). Notably, all four patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). Patients with detectable circulating tumor DNA had an inferior disease free survival (p < 0.0001; median disease-free survival: 4.6 mos. vs. not reached; hazard ratio = 12.6, 95% confidence interval: 3.06-52.2). Our study shows that next-generation circulating tumor DNA sequencing of triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy can predict recurrence with high specificity, but moderate sensitivity. For those patients where circulating tumor DNA is detected, recurrence is rapid

Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple negative breast cancer

Triple negative breast cancer accounts for 15-20% of all breast cancer cases, but despite its lower incidence, contributes to a disproportionately higher rate of mortality. As there are currently no Food and Drug Administration-approved targeted agents for triple negative breast cancer, we embarked on a genomic-guided effort to identify novel targeted modalities. Analyses by our group and The Cancer Genome Atlas have identified activation of the PI3K-pathway in the majority of triple negative breast cancers. As single agent therapy is commonly subject to resistance, we investigated the use of combination therapy against compensatory pathways. Herein, we demonstrate that pan-PI3K inhibition in triple negative breast cancers results in marked activation of the Wnt-pathway. Using the combination of two inhibitors currently in clinical trial as single agents, buparlisib(pan-PI3K) and WNT974(WNT-pathway), we demonstrate significant in vitro and in vivo synergy against triple negative breast cancer cell lines and xenografts. Taken together, these observations provide a strong rationale for testing dual targeting of the PI3K and WNT-pathways in clinical trials

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas

BACKGROUND: Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing. METHODS: The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used. RESULTS: Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability. CONCLUSIONS: A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855)

Whole-Genome Sequencing to Identify the Genetic Etiology of a Spontaneous Thymoma Mouse Model

Background: A mouse model for thymoma was previously created serendipitously by the random introduction of a transgene consisting of a mouse α-cardiac promoter, a constitutively active human transforming growth factor-β, and a simian virus 40 integration sequence into C3HeB/FeJ mice. Previous data demonstrated that the likely cause of thymomas in the thymoma mouse model was due to insertional mutagenesis by the transgene. At the time, fluorescence in situ hybridization was used to localize the transgene to the short arm of chromosome 2 (Chr2qF2-G region). In this exploratory study, we aimed to identify the exact insertion site of the transgene as this could provide clues to the genetic causation of thymomas in humans. Materials and Methods: To identify the insertion site of the transgene, germline DNA from the thymoma mouse model was sequenced using low-pass, fragment-library, whole genome sequencing. Long-insert mate pair whole genome sequencing was employed to traverse the repetitive regions of the mouse’s genome and identify the integration site. Results: The transgene was found to be integrated into a repetitive area of the mouse genome, specifically on Chr2qF1 within the intron of the FAM227B gene. Tandem integration of the transgene was observed with enumeration of an estimated 30 copies. Initial results suggested that a nearby gene, fibroblast growth factor 7 (Fgf7), could be affected by the gene insertion. Conclusions: Whole genome sequencing of this thymoma mouse model identified the region of tandem integration of a transgene on Chr2qF1 that may have potential translational implications in helping to understand the genomic etiology of thymoma in humans

Characterizing the heterogeneity of triple-negative breast cancers using microdissected normal ductal epithelium and RNA-sequencing

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90 % of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos

Initial Phase I Safety Study of Gedatolisib plus Cofetuzumab Pelidotin for Patients with Metastatic Triple-Negative Breast Cancer

Purpose: The PI3K pathway is dysregulated in the majority of triple-negative breast cancers (TNBC), yet single-agent inhibition of PI3K has been ineffective in TNBC. PI3K inhibition leads to an immediate compensatory upregulation of the Wnt pathway. Dual targeting of both pathways is highly synergistic against TNBC models in vitro and in vivo. We initiated a phase I clinical trial combining gedatolisib, a pan-class I isoform PI3K/mTOR inhibitor, and cofetuzumab pelidotin, an antibody-drug conjugate against the cell-surface PTK7 protein (Wnt pathway coreceptor) with an auristatin payload. Patients and methods: Participants (pt) had metastatic TNBC or estrogen receptor (ER) low (ER and PgR < 5%, HER2-negative) breast cancer, and had received at least one prior chemotherapy for advanced disease. The primary objective was safety. Secondary endpoints included overall response rate (ORR), clinical benefit at 18 weeks (CB18), progression-free survival (PFS), and correlative analyses. Results: A total of 18 pts were enrolled in three dose cohorts: gedatolisib 110 mg weekly + cofetuzumab pelidotin 1.4 mg/kg every 3 weeks (n = 4), 180 mg + 1.4 mg/kg (n = 3), and 180 mg + 2.8 mg/kg (n = 11). Nausea, anorexia, fatigue, and mucositis were common but rarely reached ≥grade 3 severity. Myelosuppression was uncommon. ORR was 16.7% (3/18). An additional 3 pts had stable disease (of these 2 had stable disease for >18 weeks); CB18 was 27.8%. Median PFS was 2.0 months (95% confidence interval for PFS: 1.2-6.2). Pts with clinical benefit were enriched with genomic alterations in the PI3K and PTK7 pathways. Conclusions: The combination of gedatolisib + cofetuzumab pelidotin was well tolerated and demonstrated promising clinical activity. Further investigation of this drug combination in metastatic TNBC is warranted

Molecular characterization of the translocation breakpoints in the Down syndrome mouse model Ts65Dn.

Ts65Dn is a mouse model of Down syndrome: a syndrome that results from chromosome (Chr) 21 trisomy and is associated with congenital defects, cognitive impairment, and ultimately Alzheimer\u27s disease. Ts65Dn mice have segmental trisomy for distal mouse Chr 16, a region sharing conserved synteny with human Chr 21. As a result, this strain harbors three copies of over half of the human Chr 21 orthologs. The trisomic segment of Chr 16 is present as a translocation chromosome (Mmu17(16)), with breakpoints that have not been defined previously. To molecularly characterize the Chrs 16 and 17 breakpoints on the translocation chromosome in Ts65Dn mice, we used a selective enrichment and high-throughput paired-end sequencing approach. Analysis of paired-end reads flanking the Chr 16, Chr 17 junction on Mmu17(16) and de novo assembly of the reads directly spanning the junction provided the precise locations of the Chrs 16 and 17 breakpoints at 84,351,351 and 9,426,822 bp, respectively. These data provide the basis for low-cost, highly efficient genotyping of Ts65Dn mice. More importantly, these data provide, for the first time, complete characterization of gene dosage in Ts65Dn mice

Profiling molecular regulators of recurrence in chemorefractory triple-negative breast cancers

BACKGROUND: Approximately two thirds of patients with localized triple-negative breast cancer (TNBC) harbor residual disease (RD) after neoadjuvant chemotherapy (NAC) and have a high risk-of-recurrence. Targeted therapeutic development for TNBC is of primary significance as no targeted therapies are clinically indicated for this aggressive subset. In view of this, we conducted a comprehensive molecular analysis and correlated molecular features of chemorefractory RD tumors with recurrence for the purpose of guiding downstream therapeutic development. METHODS: We assembled DNA and RNA sequencing data from RD tumors as well as pre-operative biopsies, lymphocytic infiltrate, and survival data as part of a molecular correlative to a phase II post-neoadjuvant clinical trial. Matched somatic mutation, gene expression, and lymphocytic infiltrate were assessed before and after chemotherapy to understand how tumors evolve during chemotherapy. Kaplan-Meier survival analyses were conducted categorizing cancers with TP53 mutations by the degree of loss as well as by the copy number of a locus of 18q corresponding to the SMAD2, SMAD4, and SMAD7 genes. RESULTS: Analysis of matched somatic genomes pre-/post-NAC revealed chaotic acquisition of copy gains and losses including amplification of prominent oncogenes. In contrast, significant gains in deleterious point mutations and insertion/deletions were not observed. No trends between clonal evolution and recurrence were identified. Gene expression data from paired biopsies revealed enrichment of actionable regulators of stem cell-like behavior and depletion of immune signaling, which was corroborated by total lymphocytic infiltrate, but was not associated with recurrence. Novel characterization of TP53 mutation revealed prognostically relevant subgroups, which were linked to MYC-driven transcriptional amplification. Finally, somatic gains in 18q were associated with poor prognosis, likely driven by putative upregulation of TGFß signaling through the signal transducer SMAD2. CONCLUSIONS: We conclude TNBCs are dynamic during chemotherapy, demonstrating complex plasticity in subclonal diversity, stem-like qualities, and immune depletion, but somatic alterations of TP53/MYC and TGFß signaling in RD samples are prominent drivers of recurrence, representing high-yield targets for additional interrogation