An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression.

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.

Combined Simplified Molecular Classification of Gastric Adenocarcinoma, Enhanced by Lymph Node Status: An Integrative Approach.

Gastric adenocarcinoma (GAC) is a heterogeneous disease and at least two major studies have recently provided a molecular classification for this tumor: The Cancer Genome Atlas (TCGA) and the Asian Cancer Research Group (ARCG). Both classifications quote four molecular subtypes, but these subtypes only partially overlap. In addition, the classifications are based on complex and cost-intensive technologies, which are hardly feasible for everyday practice. Therefore, simplified approaches using immunohistochemistry (IHC), in situ hybridization (ISH) as well as commercially available next generation sequencing (NGS) have been considered for routine use. In the present study, we screened 115 GAC by IHC for p53, MutL Homolog 1 (MLH1) and E-cadherin and performed ISH for Epstein-Barr virus (EBV). In addition, sequencing by NGS for TP53 and tumor associated genes was performed. With this approach, we were able to define five subtypes of GAC: (1) Microsatellite Instable (MSI), (2) EBV-associated, (3) Epithelial Mesenchymal Transition (EMT)-like, (4) p53 aberrant tumors surrogating for chromosomal instability and (5) p53 proficient tumors surrogating for genomics stable cancers. Furthermore, by considering lymph node metastasis in the p53 aberrant GAC, a better prognostic stratification was achieved which finally allowed us to separate the GAC highly significant in a group with poor and good-to-intermediate prognosis, respectively. Our data show that molecular classification of GAC can be achieved by using commercially available assays including IHC, ISH and NGS. Furthermore, we present an integrative workflow, which has the potential to overcome the uncertainty resulting from discrepancies from existing classification schemes.

Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells.

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.

Single-Center Experience with a Targeted Next Generation Sequencing Assay for Assessment of Relevant Somatic Alterations in Solid Tumors.

Companion diagnostics rely on genomic testing of molecular alterations to enable effective cancer treatment. Here we report the clinical application and validation of the Oncomine Focus Assay (OFA), an integrated, commercially available next-generation sequencing (NGS) assay for the rapid and simultaneous detection of single nucleotide variants, short insertions and deletions, copy number variations, and gene rearrangements in 52 cancer genes with therapeutic relevance. Two independent patient cohorts were investigated to define the workflow, turnaround times, feasibility, and reliability of OFA targeted sequencing in clinical application and using archival material. Cohort I consisted of 59 diagnostic clinical samples from the daily routine submitted for molecular testing over a 4-month time period. Cohort II consisted of 39 archival melanoma samples that were up to 15years old. Libraries were prepared from isolated nucleic acids and sequenced on the Ion Torrent PGM sequencer. Sequencing datasets were analyzed using the Ion Reporter software. Genomic alterations were identified and validated by orthogonal conventional assays including pyrosequencing and immunohistochemistry. Sequencing results of both cohorts, including archival formalin-fixed, paraffin-embedded material stored up to 15years, were consistent with published variant frequencies. A concordance of 100% between established assays and OFA targeted NGS was observed. The OFA workflow enabled a turnaround of 3½ days. Taken together, OFA was found to be a convenient tool for fast, reliable, broadly applicable and cost-effective targeted NGS of tumor samples in routine diagnostics. Thus, OFA has strong potential to become an important asset for precision oncology.

Trametinib after disease reactivation under dabrafenib in Erdheim-Chester disease with both BRAF and KRAS mutations.

Major advances have been made in understanding the pathogenesis of Erdheim-Chester disease (ECD) leading to novel treatment strategies. Targeted therapies such as BRAF inhibition have shown a significant impact on disease management, emphasizing the importance of the activated mitogen-associated protein kinase pathway in this disease. However, incomplete responsiveness, potentially limiting adverse effects, and the occurrence of treatment resistance to BRAF inhibition observed in other BRAF-mutant malignancies imply the importance of therapeutic strategies beyond BRAF inhibition. We report a patient with ECD who carried the BRAFV600E mutation and developed treatment resistance under BRAF inhibition despite initial treatment response. Genetic analyses of a newly developing ECD lesion revealed a somatic KRASQ61H mutation without the presence of BRAFV600E Accordingly, the addition of MEK-inhibiting trametinib to BRAF-inhibiting dabrafenib was able to overcome acquired partial treatment resistance. This is the first report of treatment resistance as a result of a secondary MAPK pathway-activating mutation during BRAF inhibition in ECD. This case contributes to the ongoing efforts of simultaneous BRAF/MEK inhibition as a promising strategy in ECD.

Colorectal Choriocarcinoma in a Patient with Probable Lynch Syndrome.

BACKGROUND: Personalized therapy of colorectal cancer is influenced by morphological, molecular, and host-related factors. Here, we report the comprehensive clinicopathological and molecular analysis of an extra-gestational colorectal choriocarcinoma in a patient with probable Lynch syndrome. CASE PRESENTATION: A 61-year-old female with history of gastric cancer at age 36 presented with a transmurally invasive tumor of the right hemicolon and liver metastasis. A right hemicolectomy was performed. Histopathological analysis showed a mixed trophoblastic and syncytiotrophoblastic differentiation, consistent with choriocarcinoma. Disease progression was rapid under oxaliplatin, capecitabine, irinotecan, and bevacizumab. Molecular phenotyping identified loss of mismatch-repair protein immunostaining for PMS2, microsatellite instability, a lack of MLH1 promoter methylation, and lack of BRAF mutation suggestive of Lynch syndrome. Targeted next-generation sequencing revealed an ataxia telangiectasia mutated (p.P604S) missense mutation. A bleomycin, etoposide, and cisplatin treatment protocol targeting germ cell neoplasia lead to disease remission and prolonged survival of 34 months. CONCLUSION: Comprehensive immunohistochemical and genetic testing is essential to identify uncommon cancers possibly related to Lynch syndrome. For rare tumors, personalized therapeutic approaches should take both molecular and morphological information into account.

Chaotic neovascularization induced by aggressive fibrosarcoma cells overexpressing S-adenosylmethionine decarboxylase.

S-adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines essential for cell proliferation. Overexpression of S-adenosylmethionine decarboxylase in rodent fibroblasts led to aggressive transformants (Amdc-s cells) that had unforeseen high invasive capacity in nude mice, invading rapidly from the subcutaneous injection site into the peritoneal cavity and its organs. In vitro, these cells were much more invasive than Ras-oncogene-transformed fibroblasts, or human HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells. In immunohistological characterization, Amdc-s-induced tumors showed chaotic neovascularization, with abundant pleomorphic vessel-like structures that had noncontiguous or totally missing laminin (basement membrane) and CD31 (endothelial cell) immunoreactivity. Gene expression and protein analyses of Amdc-s cells showed them to overexpress several pro-angiogenic molecules, including vascular endothelial growth factor (VEGF-A), and to exhibit profound down-regulation of the anti-angiogenic thrombospondin-1 (TSP-1). By reintroduction of TSP-1 into Amdc-s cells, the high invasiveness was efficiently inhibited in vitro. Interestingly, Amdc-s cells showed up-regulation of hepatocyte growth factor (HGF) and also expressed the MET receptor, creating thus an autocrine loop able to regulate VEGF-A and TSP-1 levels. Further, we found Amdc-s cells to express increased amounts of matrix metalloproteinase-2 (MMP-2) and the large isoform of tenascin-C (TN-C), which may also contribute to the angiogenic switch and invasiveness. Consequently, Amdc-s cells offer an excellent model to sort out the key molecules of aggressive tumor growth, and thereby help in designing rational, novel anti-vascular and other cancer therapies.