Genomic evolutionary trajectory of metastatic squamous cell carcinoma of the lung.

BACKGROUND: The extent of inter- and intratumoral genomic heterogeneity and the clonal evolution of metastatic squamous cell carcinoma of the lung (LUSC) are poorly understood. Genomic studies of LUSC are challenged by their low tumor cell content. We sought to define the genomic landscape and evolutionary trajectories of metastatic LUSC combining nuclei-flow sorting and whole exome sequencing. METHODS: Five patients with primary LUSC and six matched metastases were investigated. Tumor nuclei were sorted based on ploidy and expression of cytokeratin to enrich for tumor cells for whole exome sequencing. RESULTS: Flow-sorting increased the mean tumor purity from 26% (range, 12-50%) to 73% (range, 42-93%). Overall, primary LUSCs and their matched metastases shared a median of 79% (range, 67-85%) of copy number aberrations (CNAs) and 74% (range, 65-94%) of non-synonymous mutations, including in tumor suppressor genes such as TP53. Furthermore, the ploidy of the tumors remained unchanged between primary and metastasis in 4/5 patients over time. We found differences in the mutational signatures of shared mutations compared to the private mutations in the primary or metastasis. CONCLUSIONS: Our results demonstrate a close genomic relationship between primary LUSCs and their matched metastases, suggesting late dissemination of the metastases from the primary tumors during tumor evolution.

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.

Novel cell enrichment technique for robust genetic analysis of archival classical Hodgkin lymphoma tissues.

Approximately 15% of patients with classical Hodgkin lymphoma (cHL) die after relapse or progressive disease. Comprehensive genetic characterization is required to better understand its molecular pathology and improve management. However, genetic information on cHL is hard to obtain mainly due to rare malignant Hodgkin- and Reed-Sternberg cells (HRSC), whose overall frequencies in the affected tissues ranges from 0.1 to 10%. Therefore, enrichment of neoplastic cells is necessary for the majority of genetic investigations. We have developed a new high-throughput method for marker-based enrichment of archival formalin-fixed and paraffin-embedded (FFPE) tissue-derived HRSC nuclei by fluorescence-assisted flow sorting (FACS) and successfully applied it on ten cHL cases. Genomic DNA extracted from sorted nuclei was used for targeted high-throughput sequencing (HTS) of 68 genes that are frequently affected in lymphomas. Chromosomal copy number aberrations were investigated by the Agilent SurePrint 180k microarray. Our method enabled HRSC nuclei enrichment to 40-90% in sorted populations. This level of enrichment was sufficient for reliable identification of tumor-specific mutations and copy number aberrations. Genetic analysis revealed that components of JAK-STAT signaling pathway were affected in all investigated tumors by frequent mutations of SOCS1 and STAT6 as well as copy number gains of JAK2. Involvement of nuclear factor-κB (NF-κB) pathway compounds was evident from recurrent gains of the locus containing the REL gene and mutations in TNFAIP3 and CARD11. Finally, genetic alterations of PD-L1 and B2M suggested immune evasion as mechanisms of oncogenesis in some patients. In this work, we present a new method for HRSC enrichment from FFPE tissue blocks by FACS and demonstrate the feasibility of a wide-scale genetic analysis by cutting-edge molecular methods. Our work opens the door to a large resource of archived clinical cHL samples and lays foundation to more complex studies aimed to answer important biological and clinical questions that are critical to improve cHL management.

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.

Extracavitary primary effusion lymphoma: clinical, morphological, phenotypic and cytogenetic characterization using nuclei enrichment technique.

AIMS: Primary effusion lymphoma (PEL) is a rare form of aggressive B-cell lymphoma, which typically manifests as malignant effusion in the body cavities. However, extracavitary solid variants are also described. The aim of this study was to investigate copy number aberrations in two cases of solid PEL at their first occurrences and relapse by applying a newly developed methodology of tumour nuclei enrichment. METHODS AND RESULTS: Using histological and genetic techniques, a novel protocol for tumour nuclei enrichment by flow sorting and array-comparative genomic hybridization, we characterized two cases of extracavitary PEL, one of which later relapsed as effusion. Both primary tumours were positive for HHV8 and EBV, confined to lymph nodes, and aberrantly expressed CD3, yet displaying clonal immunoglobulin gene rearrangements indicating B-cell origin. Cytogenetic characterization of primary tumours revealed modest number of aberrations, partially overlapping with previously reported affected loci. The effusional relapse in case 1 was cytogenetically related to the primary tumour but showed dramatic increase of chromosomal instability. CONCLUSIONS: We for the first time demonstrate a cytogenetic relationship between solid and effusional presentations of PEL. Moreover, we provide an indirect evidence of multiple malignant clones, which gave rise to clonally-related, yet karyotypically different relapsing lymphoma manifestations.

Protein phosphatase 2A promotes hepatocellular carcinogenesis in the diethylnitrosamine mouse model through inhibition of p53.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most HCCs develop in cirrhotic livers. Alcoholic liver disease, chronic hepatitis B and chronic hepatitis C are the most common underlying liver diseases. Hepatitis C virus (HCV)-specific mechanisms that contribute to HCC are presently unknown. Transgenic expression of HCV proteins in the mouse liver induces an overexpression of the protein phosphatase 2A catalytic subunit (PP2Ac). We have previously reported that HCV-induced PP2Ac overexpression modulates histone methylation and acetylation and inhibits DNA damage repair. In this study, we analyze tumor formation and gene expression using HCV transgenic mice that overexpress PP2Ac and liver tissues from patients with HCC. We demonstrate that PP2Ac overexpression interferes with p53-induced apoptosis. Injection of the carcinogen, diethylnitrosamine, induced significantly more and larger liver tumors in HCV transgenic mice that overexpress PP2Ac compared with control mice. In human liver biopsies from patients with HCC, PP2Ac expression was significantly higher in HCC tissue compared with non-tumorous liver tissue from the same patients. Our findings demonstrate an important role of PP2Ac overexpression in liver carcinogenesis and provide insights into the molecular pathogenesis of HCV-induced HCC.

Enhanced expression of ANO1 in head and neck squamous cell carcinoma causes cell migration and correlates with poor prognosis.

Head and neck squamous cell carcinoma (HNSCC) has the potential for early metastasis and is associated with poor survival. Ano1 (Dog1) is an established and sensitive marker for the diagnosis of gastrointestinal stromal tumors (GIST) and has recently been identified as a Ca(2+) activated Cl(-) channel. Although the ANO1 gene is located on the 11q13 locus, a region which is known to be amplified in different types of human carcinomas, a detailed analysis of Ano1 amplification and expression in HNSCC has not been performed. It is thus still unclear how Ano1 contributes to malignancy in HNSCC. We analyzed genomic amplification of the 11q13 locus and Ano1 together with Ano1-protein expression in a large collection of HNSCC samples. We detected a highly significant correlation between amplification and expression of Ano1 and showed that HNSCC patients with Ano1 protein expression have a poor overall survival. We further analyzed the expression of the Ano1 protein in more than 4'000 human samples from 80 different tumor types and 76 normal tissue types and detected that besides HNSCC and GISTs, Ano1 was rarely expressed in other tumor samples or healthy human tissues. In HNSCC cell lines, expression of Ano1 caused Ca(2+) activated Cl(-) currents, which induced cell motility and cell migration in wound healing and in real time migration assays, respectively. In contrast, knockdown of Ano1 did not affect intracellular Ca(2+) signaling and surprisingly did not reduce cell proliferation in BHY cells. Further, expression and activity of Ano1 strongly correlated with the ability of HNSCC cells to regulate their volume. Thus, poor survival in HNSCC patients is correlated with the presence of Ano1. Our results further suggest that Ano1 facilitates regulation of the cell volume and causes cell migration, which both can contribute to metastatic progression in HNSCC.