Experimental and spontaneous metastasis assays can result in divergence in clonal architecture.

Intratumoural heterogeneity is associated with poor outcomes in breast cancer. To understand how malignant clones survive and grow in metastatic niches, in vivo models using cell lines and patient-derived xenografts (PDX) have become the gold standard. Injections of cancer cells in orthotopic sites (spontaneous metastasis assays) or into the vasculature (experimental metastasis assays) have been used interchangeably to study the metastatic cascade from early events or post-intravasation, respectively. However, less is known about how these different routes of injection impact heterogeneity. Herein we directly compared the clonality of spontaneous and experimental metastatic assays using the human cell line MDA-MB-231 and a PDX model. Genetic barcoding was used to study the fitness of the subclones in primary and metastatic sites. Using spontaneous assays, we found that intraductal injections resulted in less diverse tumours compared to other routes of injections. Using experimental metastasis assays via tail vein injection of barcoded MDA-MB-231 cells, we also observed an asymmetry in metastatic heterogeneity between lung and liver that was not observed using spontaneous metastasis assays. These results demonstrate that these assays can result in divergent clonal outputs in terms of metastatic heterogeneity and provide a better understanding of the biases inherent to each technique.

Epigenetic modulators of B cell fate identified through coupled phenotype-transcriptome analysis.

High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.

Survival Following Chemotherapy in Ovarian Clear Cell Carcinoma Is Not Associated with Pathological Misclassification of Tumor Histotype.

PURPOSE: Although ovarian clear cell carcinomas (OCCC) are commonly resistant to platinum-based chemotherapy, good clinical outcomes are observed in a subset of patients. The explanation for this is unknown but may be due to misclassification of high-grade serous ovarian cancer (HGSOC) as OCCC or mixed histology. EXPERIMENTAL DESIGN: To discover potential biomarkers of survival benefit following platinum-based chemotherapy, we ascertained a cohort of 68 Japanese and Australian patients in whom progression-free survival (PFS) and overall survival (OS) could be assessed. We performed IHC reclassification of tumors, and targeted sequencing and immunohistochemistry of known driver genes. Exome sequencing was performed in 10 patients who had either unusually long survival (N = 5) or had a very short time to progression (N = 5). RESULTS: The majority of mixed OCCC (N = 6, 85.7%) and a small proportion of pure OCCC (N = 3, 4.9%) were reclassified as likely HGSOC. However, the PFS and OS of patients with misclassified samples were similar to that of patients with pathologically validated OCCC. Absent HNF1B expression was significantly correlated with longer PFS and OS (P = 0.0194 and 0.0395, respectively). Mutations in ARID1A, PIK3CA, PPP2R1A, and TP53 were frequent, but did not explain length of PFS and OS. An exploratory exome analysis of patients with favorable and unfavorable outcomes did not identify novel outcome-associated driver mutations. CONCLUSIONS: Survival benefit following chemotherapy in OCCC was not associated with pathological misclassification of tumor histotype. HNF1B loss may help identify the subset of patients with OCCC with a more favorable outcome.

Homologous Recombination DNA Repair Pathway Disruption and Retinoblastoma Protein Loss Are Associated with Exceptional Survival in High-Grade Serous Ovarian Cancer.

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths.Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing.Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival.Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569-80. ©2017 AACRSee related commentary by Peng and Mills, p. 508.

Selective enrichment of STRs for applications in forensic human identification.

Forensic human identification (HID) is currently based on determining repeat length polymorphisms located in short tandem repeat regions in the human genome. Despite the great progress made in the area of multiplex PCR-based approaches, limitations associated with challenging forensic samples such as DNA degradation, cooccurrence of inhabited microbial DNA and PCR inhibitors significantly affect the success rate of human DNA profiling. We have developed a sequence-specific pre-PCR STR enrichment method and evaluated its efficacy using DNA samples doped with various contaminants in view of its application on compromised forensic samples. This strategy has enabled us to generate complete and reproducible DNA profiles from samples doped with fivefold excess of nonhuman DNA and three to fourfold excess of various potent PCR inhibitors than that is claimed to be tolerated by some of the widely used commercial multiplex STR kits, from as little as two nanograms of degraded human DNA. The "hybrid capture"-based STR enrichment strategy described in this study is easily adaptable and offers a sensitive, efficient, and economical approach for successful human DNA profiling from compromised and recalcitrant forensic samples that are usually encountered in mass disaster incidents and missing persons' identifications.