Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

BACKGROUND: Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. RESULTS: To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. CONCLUSIONS: Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.

Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Synergistic combination of cytotoxic chemotherapy and cyclin-dependent kinase 4/6 inhibitors in biliary tract cancers.

BACKGROUND AND AIMS: Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin-dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive. APPROACH AND RESULTS: Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy-induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine. CONCLUSIONS: As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

Genomic landscape of Epstein-Barr virus-positive extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue.

Epstein-Barr virus (EBV)-positive extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT lymphomas) were initially described in solid organ transplant recipients, and, more recently, in other immunodeficiency settings. The overall prevalence of EBV-positive MALT lymphomas has not been established, and little is known with respect to their genomic characteristics. Eight EBV-positive MALT lymphomas were identified, including 1 case found after screening a series of 88 consecutive MALT lymphomas with EBER in situ hybridization (1%). The genomic landscape was assessed in 7 of the 8 cases with a targeted high throughput sequencing panel and array comparative genomic hybridization. Results were compared to published data for MALT lymphomas. Of the 8 cases, 6 occurred post-transplant, 1 in the setting of primary immunodeficiency, and 1 case was age-related. Single pathogenic/likely pathogenic mutations were identified in 4 of 7 cases, including mutations in IRF8, BRAF, TNFAIP3, and SMARCA4. Other than TNFAIP3, these genes are mutated in <3% of EBV-negative MALT lymphomas. Copy number abnormalities were identified in 6 of 7 cases with a median of 6 gains and 2 losses per case, including 4 cases with gains in regions encompassing several IRF family or interacting genes (IRF2BP2, IRF2, and IRF4). There was no evidence of trisomies of chromosomes 3 or 18. In summary, EBV-positive MALT lymphomas are rare and, like other MALT lymphomas, are usually genetically non-complex. Conversely, while EBV-negative MALT lymphomas typically show mutational abnormalities in the NF-κB pathway, other than the 1 TNFAIP3-mutated case, no other NF-κB pathway mutations were identified in the EBV-positive cases. EBV-positive MALT lymphomas often have either mutations or copy number abnormalities in IRF family or interacting genes, suggesting that this pathway may play a role in these lymphomas.

Exploring the spatiotemporal genetic heterogeneity in metastatic lung adenocarcinoma using a nuclei flow-sorting approach.

Variable tumor cellularity can limit sensitivity and precision in comparative genomics because differences in tumor content can result in misclassifying truncal mutations as region-specific private mutations in stroma-rich regions, especially when studying tissue specimens of mediocre tumor cellularity such as lung adenocarcinomas (LUADs). To address this issue, we refined a nuclei flow-sorting approach by sorting nuclei based on ploidy and the LUAD lineage marker thyroid transcription factor 1 and applied this method to investigate genome-wide somatic copy number aberrations (SCNAs) and mutations of 409 cancer genes in 39 tumor populations obtained from 16 primary tumors and 21 matched metastases. This approach increased the mean tumor purity from 54% (range 7-89%) of unsorted material to 92% (range 79-99%) after sorting. Despite this rise in tumor purity, we detected limited genetic heterogeneity between primary tumors and their metastases. In fact, 88% of SCNAs and 80% of mutations were propagated from primary tumors to metastases and low allele frequency mutations accounted for much of the mutational heterogeneity. Even though the presence of SCNAs indicated a history of chromosomal instability (CIN) in all tumors, metastases did not have more SCNAs than primary tumors. Moreover, tumors with biallelic TP53 or ATM mutations had high numbers of SCNAs, yet they were associated with a low interlesional genetic heterogeneity. The results of our study thus provide evidence that most macroevolutionary events occur in primary tumors before metastatic dissemination and advocate for a limited degree of CIN over time and space in this cohort of LUADs. Sampling of primary tumors thus may suffice to detect most mutations and SCNAs. In addition, metastases but not primary tumors had seeded additional metastases in three of four patients; this provides a genomic rational for surgical treatment of such oligometastatic LUADs. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Enhanced DNA repair and genomic stability identify a novel HIV-related diffuse large B-cell lymphoma signature.

Diffuse large B-cell lymphoma (DLBCL) is up to 17-fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (-) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(-) germinal center B-cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression (CCNA2, CCNB1, CDC25A, E2F1), DNA replication (MCM2, MCM4, MCM7) and DNA damage repair, including eight Fanconi anemia genes (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT/UBE2T, FANCV/MAD2L2), were significantly increased in HIV(+) GCB-DLBCL tumors compared to HIV(-) tumors. In contrast, genes associated with cell cycle inhibition (CDKN1A, CDKN1B) as well as apoptosis regulating BCL2 family members (BCL2, BAX, BIM, BMF, PUMA) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB-DLBCL tumors have fewer copy number variations than their HIV(-) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB-DLBCL is a distinct molecular malignancy from HIV(-) GCB-DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.

Delineation of human prostate cancer evolution identifies chromothripsis as a polyclonal event and FKBP4 as a potential driver of castration resistance.

Understanding the evolutionary mechanisms and genomic events leading to castration-resistant (CR) prostate cancer (PC) is key to improve the outcome of this otherwise deadly disease. Here, we delineated the tumour history of seven patients progressing to castration resistance by analysing matched prostate cancer tissues before and after castration. We performed genomic profiling of DNA content-based flow-sorted populations in order to define the different evolutionary patterns. In one patient, we discovered that a catastrophic genomic event, known as chromothripsis, resulted in multiple CRPC tumour populations with distinct, potentially advantageous copy number aberrations, including an amplification of FK506 binding protein 4 (FKBP4, also known as FKBP52), a protein enhancing the transcriptional activity of androgen receptor signalling. Analysis of FKBP4 protein expression in more than 500 prostate cancer samples revealed increased expression in CRPC in comparison to hormone-naïve (HN) PC. Moreover, elevated FKBP4 expression was associated with poor survival of patients with HNPC. We propose FKBP4 amplification and overexpression as a selective advantage in the process of tumour evolution and as a potential mechanism associated with the development of CRPC. Furthermore, FKBP4 interaction with androgen receptor may provide a potential therapeutic target in PC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The association of genomic lesions and PD-1/PD-L1 expression in resected triple-negative breast cancers.

BACKGROUND: Elevated PD-L1 expression on tumor cells, a context associated with an adaptive immune response, has been linked to the total burden of copy number variants (CNVs) in aneuploid tumors, to microsatellite instability (MSI), and to specific genomic driver lesions, including loss of PTEN, MYC amplification, and activating mutations in driver oncogenes such as KRAS and PIK3CA. Triple-negative breast cancers (TNBCs) typically have high levels of CNVs and diverse driver lesions in their genomes. Thus, there is significant interest in exploiting genomic data to develop predictive immunotherapy biomarkers for patients with TNBC. METHODS: Whole tissue samples from 55 resected TNBCs were screened by immunohistochemistry (IHC) for PD-1 and PD-L1 by using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. In parallel, we interrogated biopsies from each resection with DNA content flow cytometry and sorted the nuclei of diploid, tetraploid, and aneuploid cell populations. CNVs were mapped with CNV oligonucleotide arrays by using purified (>95%) tumor populations. We generated whole exome data for 12 sorted tumor samples to increase the resolution within loci of interest and to incorporate somatic mutations into our genomic signatures. RESULTS AND CONCLUSIONS: PD-L1 staining was detected on tumor cells in 29 out of 54 (54%) evaluable cases and was associated with increased overall survival (P = 0.0024). High levels of PD-1 and PD-L1 (IHC ≥4) were present in 11 out of 54 (20%) and 20 out of 54 (37%) cases with staining of PD-L1 primarily on tumor cells for 17 out of 20 (85%) cases. The latter included tumors with both high (>50) and low (<20) numbers of CNVs. Notably, homozygous deletion of PTEN (n = 6) or activating mutation in PIK3CA (n = 1) was not associated with increased expression of either immune checkpoint activator in TNBC. In contrast, two treatment-naïve cases with EGFR driver amplicons had high PD-L1 tumor staining. High mutational load and predicted neoepitopes were observed in MSI+ and high CNV burden TNBCs but were not associated with high PD-L1 expression on tumor cells. Our results challenge current models of genomic-based immunotherapy signatures yet suggest that discrete genomic lesions may complement existing biomarkers to advance immune checkpoint therapies for patients with TNBC.

Clinical study of genomic drivers in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials. METHODS: Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed. RESULTS: We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its' 3' region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection. CONCLUSIONS: We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.

Development and validation of a novel clinical fluorescence in situ hybridization assay to detect JAK2 and PD-L1 amplification: a fluorescence in situ hybridization assay for JAK2 and PD-L1 amplification.

The amplification of chromosome 9p24.1 encoding PD-L1, PD-L2, and JAK2 has been reported in multiple types of cancer and is associated with poor outcome, upregulation of PD-L1, and activation of the JAK/STAT pathway. We have developed a novel fluorescence in situ hybridization assay which combines 3 probes mapping to 9p24.1 with a commercial chromosome 9 centromere (CEN9) probe for detection of the JAK2/9p24.1 amplification. JAK2 fluorescence in situ hybridization was compared with array-based comparative genomic hybridization in 34 samples of triple negative breast cancer tumor. By array-based comparative genomic hybridization, 15 had 9p24.1 copy-number gain (log2ratio>0.3) and 19 were classified as non-gain (log2ratio≤0.3). Copy-number gain was defined as JAK2/CEN9 ratio ≥1.1 or average JAK2 signals≥3.0. Twelve of 15 samples with copy-number gain by array-based comparative genomic hybridization were also detected by fluorescence in situ hybridization. Eighteen of 19 samples classified as copy-number non-gain by array-based comparative genomic hybridization were concordant by array-based comparative genomic hybridization. The sensitivity and specificity of the fluorescence in situ hybridization assay was 80% and 95%, respectively (P=0.02). The sample with the highest level of amplification by array-based comparative genomic hybridization (log2ratio=3.6) also scored highest by fluorescence in situ hybridization (ratio=8.2). There was a correlation between the expression of JAK2 and amplification status (Mean 633 vs 393, P=0.02), and there was a trend of association with PD-L1 RNA expression (Mean 46 vs 22, P=0.11). No significant association was observed between PD-L1 immunohistochemistry expression and copy-number gain status. In summary, the novel array-based comparative genomic hybridization assay for detection of chromosome 9p24.1 strongly correlates with the detection of copy-number gain by array-based comparative genomic hybridization. In triple negative breast cancer, this biomarker may identify a relevant subset of patients for targeted molecular therapies.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Chromosome-breakage genomic instability and chromothripsis in breast cancer.

BACKGROUND: Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome. RESULTS: We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers. CONCLUSIONS: Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Androgen receptor-positive triple negative breast cancer: a unique breast cancer subtype.

BACKGROUND: The significance of androgen receptor (AR) expression in triple-negative breast cancer (TNBC) is unclear, and published studies so far have been inconclusive. METHODS: A tissue microarray was constructed using tissue obtained from 119 patients with primary TNBC and stained for AR expression. Other tissue types obtained included recurrent TNBC, normal breast tissue, adjacent ductal carcinoma-in situ (DCIS), lymph node (LN) and distant metastases. Positive AR expression was defined as ≥10% nuclear staining. RESULTS: Epithelial tissue was present and evaluable in 94 TNBC patients with a total of 177 tissue cores. AR expression in TNBC was 22 of 94 (23%). AR expression was higher in normal breast tissue (88%) and adjacent DCIS (73% overall). All LN metastases from AR-positive TNBC patients were also AR positive; in addition, no AR-negative TNBC patient had AR-positive LNs. AR expression was associated with older patient age (63 vs. 57 years, respectively, p = 0.051) and LN metastases (p = 0.033). Locoregional recurrence and overall/disease-specific survival were similar between AR-positive and AR-negative patients, although AR-positive patients had more advanced disease. On multivariate analysis, the presence of LN metastases was associated with poorer recurrence-free survival in AR-positive patients (hazard ratio, 4.34) (p = 0.031). CONCLUSIONS: The AR is expressed in normal breast tissue, and expression decreases with advancement to DCIS and invasive cancer. AR-positive TNBC was more common in older patients and had a higher propensity for LN metastases. AR-positive TNBC may represent a breast cancer subtype with unique features that may be amenable to treatment with alternative targeted therapies.

Glioblastomas are composed of genetically divergent clones with distinct tumourigenic potential and variable stem cell-associated phenotypes.

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM.

Advancing a clinically relevant perspective of the clonal nature of cancer.

Cancers frequently arise as a result of an acquired genomic instability and the subsequent clonal evolution of neoplastic cells with variable patterns of genetic aberrations. Thus, the presence and behaviors of distinct clonal populations in each patient's tumor may underlie multiple clinical phenotypes in cancers. We applied DNA content-based flow sorting to identify and isolate the nuclei of clonal populations from tumor biopsies, which was coupled with array CGH and targeted resequencing. The results produced high-definition genomic profiles of clonal populations from 40 pancreatic adenocarcinomas and a set of prostate adenocarcinomas, including serial biopsies from a patient who progressed to androgen-independent metastatic disease. The genomes of clonal populations were found to have patient-specific aberrations of clinical relevance. Furthermore, we identified genomic aberrations specific to therapeutically responsive and resistant clones arising during the evolution of androgen-independent metastatic prostate adenocarcinoma. We also distinguished divergent clonal populations within single biopsies and mapped aberrations in multiple aneuploid populations arising in primary and metastatic pancreatic adenocarcinoma. We propose that our high-definition analyses of the genomes of distinct clonal populations of cancer cells in patients in vivo can help guide diagnoses and tailor approaches to personalized treatment.

Genomic landscape of diploid and aneuploid microsatellite stable early onset colorectal cancer.

Although colorectal cancer (CRC) remains the second leading cause of cancer-related death in the United States, the overall incidence and mortality from the disease have declined in recent decades. In contrast, there has been a steady increase in the incidence of CRC in individuals under 50 years of age. Hereditary syndromes contribute disproportionately to early onset CRC (EOCRC). These include microsatellite instability high (MSI+) tumors arising in patients with Lynch Syndrome. However, most EOCRCs are not associated with familial syndromes or MSI+ genotypes. Comprehensive genomic profiling has provided the basis of improved more personalized treatments for older CRC patients. However, less is known about the basis of sporadic EOCRC. To define the genomic landscape of EOCRC we used DNA content flow sorting to isolate diploid and aneuploid tumor fractions from 21 non-hereditary cases. We then generated whole exome mutational profiles for each case and whole genome copy number, telomere length, and EGFR immunohistochemistry (IHC) analyses on subsets of samples. These results discriminate the molecular features of diploid and aneuploid EOCRC and provide a basis for larger population-based studies and the development of effective strategies to monitor and treat this emerging disease.

Patient-Derived Tumor Xenograft Study with CDK4/6 Inhibitor Plus AKT Inhibitor for the Management of Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive malignancy with poor outcomes. To investigate novel therapeutic strategies, we characterized three new metastatic prostate cancer patient derived-tumor xenograft (PDTX) models and developed 3D spheroids from each to investigate molecular targeted therapy combinations including CDK4/6 inhibitors (CDK4/6i) with AKT inhibitors (ATKi). Metastatic prostate cancer tissue was collected and three PDTX models were established and characterized using whole-exome sequencing. PDTX 3D spheroids were developed from these three PDTXs to show resistance patterns and test novel molecular-targeted therapies. CDK4/6i's were combined with AKTi's to assess synergistic antitumor response to prove our hypothesis that blockade of AKT overcomes drug resistance to CDK4/6i. This combination was evaluated in PDTX three-dimensional (3D) spheroids and in vivo experiments with responses measured by tumor volumes, PSA, and Ga-68 PSMA-11 PET-CT imaging. We demonstrated CDK4/6i's with AKTi's possess synergistic antitumor activity in three mCRPC PDTX models. These models have multiple unique pathogenic and deleterious genomic alterations with resistance to single-agent CDK4/6i's. Despite this, combination therapy with AKTi's was able to overcome resistance mechanisms. The IHC and Western blot analysis confirmed on target effects, whereas tumor volume, serum PSA ELISA, and radionuclide imaging demonstrated response to therapy with statistically significant SUV differences seen with Ga-68 PSMA-11 PET-CT. These preclinical data demonstrating antitumor synergy by overcoming single-agent CDK 4/6i as well as AKTi drug resistance provide the rational for a clinical trial combining a CDK4/6i with an AKTi in patients with mCRPC whose tumor expresses wild-type retinoblastoma 1.

A platform for dissecting force sensitivity and multivalency in actin networks.

The physical structure and dynamics of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them into specific functional architectures. Mechanical force has been shown to modulate the interactions between several ABPs and individual actin filaments, but it is unclear how this phenomenon contributes to the emergent force-responsive functional dynamics of actin networks. Here, we engineer filament linker complexes and combine them with photo-micropatterning of myosin motor proteins to produce an in vitro reconstitution platform for examining how force impacts the behavior of ABPs within multi-filament assemblies. Our system enables the monitoring of dozens of actin networks with varying architectures simultaneously using total internal reflection fluorescence microscopy, facilitating detailed dissection of the interplay between force-modulated ABP binding and network geometry. We apply our system to study a dimeric form of the critical cell-cell adhesion protein α-catenin, a model force-sensitive ABP. We find that myosin forces increase α-catenin's engagement of small filament bundles embedded within networks. This activity is absent in a force-sensing deficient mutant, whose binding scales linearly with bundle size in both the presence and absence of force. These data are consistent with filaments in smaller bundles bearing greater per-filament loads that enhance α-catenin binding, a mechanism that could equalize α-catenin's distribution across actin-myosin networks of varying sizes in cells to regularize their stability and composition.

Multi-modal efficacy of a chimeric vesiculovirus expressing the Morreton glycoprotein in sarcoma.

Vesiculoviruses are attractive oncolytic virus platforms due to their rapid replication, appreciable transgene capacity, broad tropism, limited preexisting immunity, and tumor selectivity through type I interferon response defects in malignant cells. We developed a synthetic chimeric virus (VMG) expressing the glycoprotein (G) from Morreton virus (MorV) and utilizing the remaining structural genes from vesicular stomatitis virus (VSV). VMG exhibited in vitro efficacy by inducing oncolysis in a broad range of sarcoma subtypes across multiple species. Notably, all cell lines tested showed the ability of VMG to yield productive infection with rapid replication kinetics and induction of apoptosis. Furthermore, pilot safety evaluations of VMG in immunocompetent, non-tumor-bearing mice showed an absence of toxicity with intranasal doses as high as 1e10 50% tissue culture infectious dose (TCID50)/kg. Locoregional administration of VMG in vivo resulted in tumor reduction in an immunodeficient Ewing sarcoma xenograft at doses as low as 2e5 TCID50. In a murine syngeneic fibrosarcoma model, while no tumor inhibition was achieved with VMG, there was a robust induction of CD8+ T cells within the tumor. The studies described herein establish the promising potential for VMG to be used as a novel oncolytic virotherapy platform with anticancer effects in sarcoma.

PDJ amplicon in triple negative breast cancer.

Amplification of chromosome 9p24.1 targeting PD-L1, PD-L2, and JAK2 (PDJ amplicon) is present in subsets of triple negative breast cancers (TNBCs) and is associated with poor clinical outcomes. However, the prevalence of PDJ+ TNBCs varies extensively across studies applying different methods for interrogating samples of interest. To rigorously assess the prevalence of PDJ amplicons in TNBC, its prognostic value and whether it is enriched by chemotherapy, we interrogated 360 TNBC samples including 74 surgical resections from patients treated in the neoadjuvant setting, and tissue microarrays (TMAs) with 31 cases from African American women and 255 resected non-metastatic cases, with a 3 color fluorescence in situ hybridization (FISH) assay targeting the 9p24.1 PDJ amplicon, 9q24.3, and 9q34.1. Samples with mean PDJ signal of > 4.5 copies, and ratios of PDJ/9q24 ≥ 2 and/or PDJ/9q34.1 ≥ 2 were called amplified (PDJ+). Correlative analyses included the association of tumor infiltrating lymphocytes (TILs) with PDJ amplicons in TNBCs. In addition, we investigated intratumor copy number of PDJ amplicons in PDJ+ and PDJ- TNBCs. Matched pre- and post-neoadjuvant treatment biopsies were available from patients (n = 6) to evaluate the effects of therapy on PDJ status. Our study provides a rigorous analysis of the prevalence, distribution, and clinical correlatives of the PDJ amplicon in TNBC.