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.

Retrospective Post-mortem SARS-CoV-2 RT-PCR of Autopsies with COVID-19-Suggestive Pathology Supports the Absence of Lethal Community Spread in Basel, Switzerland, before February 2020.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread around the world. While the first case was recorded in Hubei in December 2019, the extent of early community spread in Central Europe before this period is unknown. A high proportion of asymptomatic cases and undocumented infections, high transmissibility, and phylogenetic genomic diversity have engendered the controversial possibility of early international community spread of SARS-CoV-2 before its emergence in China. METHODS: To assess the early presence of lethal COVID-19 in Switzerland, we retrospectively performed an analysis of deaths at University Hospital Basel between October 2019 and February 2020 (n = 310), comparing the incidence of clinical causes of death with March 2020 (n = 72), the month during which the first lethal COVID-19 cases in Basel were reported. Trends of COVID-19-suggestive sequelae, such as bronchopneumonia with organization, acute respiratory distress syndrome (ARDS), or pulmonary embolisms (PE) were evaluated. In cases where autopsy was performed (n = 71), analogous analyses were conducted on the cause of death and pulmonary histological findings. Eight cases with a COVID-19-suggestive clinical history and histopathology between October 2019 and February 2020, and 3 cases before October 2019, were selected for SARS-CoV-2 RT-PCR. RESULTS: A statistically significant rise in pulmonary causes of death was observed in March 2020 (p = 0.03), consistent with the reported emergence of lethal COVID-19 in Switzerland. A rise in lethal bronchopneumonia was observed between December 2019 and January 2020, which was likely seasonal. The incidence of lethal ARDS and PE was uniformly low between October 2019 and February 2020. All autopsy cases analyzed by means of SARS-CoV-2 RT-PCR yielded negative results. CONCLUSION: Our data suggest the absence of early lethal community spread of COVID-19 in Basel before its initial reported emergence in Switzerland in March 2020.

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.

Diagnostic Targeted Sequencing Panel for Hepatocellular Carcinoma Genomic Screening.

Commercially available targeted panels miss genomic regions frequently altered in hepatocellular carcinoma (HCC). We sought to design and benchmark a sequencing assay for genomic screening of HCC. We designed an AmpliSeq custom panel targeting all exons of 33 protein-coding and two long noncoding RNA genes frequently mutated in HCC, TERT promoter, and nine genes with frequent copy number alterations. By using this panel, the profiling of DNA from fresh-frozen (n = 10, 1495×) and/or formalin-fixed, paraffin-embedded (FFPE) tumors with low-input DNA (n = 36, 530×) from 39 HCCs identified at least one somatic mutation in 90% of the cases. Median of 2.5 (range, 0 to 74) and 3 (range, 0 to 76) mutations were identified in fresh-frozen and FFPE tumors, respectively. Benchmarked against the mutations identified from Illumina whole-exome sequencing (WES) of the corresponding fresh-frozen tumors (105×), 98% (61 of 62) and 100% (104 of 104) of the mutations from WES were detected in the 10 fresh-frozen tumors and the 36 FFPE tumors, respectively, using the HCC panel. In addition, 18 and 70 somatic mutations in coding and noncoding genes, respectively, not found by WES were identified by using our HCC panel. Copy number alterations between WES and our HCC panel showed an overall concordance of 86%. In conclusion, we established a cost-effective assay for the detection of genomic alterations in HCC.

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.

Donor-derived, metastatic urothelial cancer after kidney transplantation associated with a potentially oncogenic BK polyomavirus.

BK polyomavirus has been linked to urothelial carcinoma in immunosuppressed patients. Here, we performed comprehensive genomic analysis of a BK polyomavirus-associated, metachronous, multifocal and metastatic micropapillary urothelial cancer in a kidney transplant recipient. Dissecting cancer heterogeneity by sorting technologies prior to array-comparative genomic hybridization followed by short tandem repeat analysis revealed that the metastatic urothelial cancer was of donor origin (4-year-old male). The top 50 cancer-associated genes showed no key driver mutations as assessed by next-generation sequencing. Whole genome sequencing and BK polyomavirus-specific amplification provided evidence for episomal and subgenomic chromosomally integrated BK polyomavirus genomes, which carried the same unique 17-bp deletion signature in the viral non-coding control region (NCCR). Whereas no role in oncogenesis could be attributed to the host gene integration in chromosome 1, the 17-bp deletion in the NCCR increased early viral gene expression, but decreased viral replication capacity. Consequently, urothelial cells were exposed to high levels of the transforming BK polyomavirus early proteins large tumour antigen and small tumour antigen from episomal and integrated gene expression. Surgery combined with discontinuation of immunosuppression resulted in complete remission, but sacrificed the renal transplant. Thus, this report links, for the first time, BK polyomavirus NCCR rearrangements with oncogenic transformation in urothelial cancer in immunosuppressed patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Vascular endothelial growth factor A amplification in colorectal cancer is associated with reduced M1 and M2 macrophages and diminished PD-1-expressing lymphocytes.

VEGFA is an angiogenic factor secreted by tumors, in particular those with VEGFA amplification, as well as by macrophages and lymphocytes in the tumor microenvironment. Here we sought to define the presence of M1/M2 macrophages, PD-1-positive lymphocytes and PD-L1 tumoral and stromal expression in colorectal cancers harboring VEGFA amplification or chromosome 6 polysomy. 38 CRCs of which 13 harbored VEGFA amplification, 6 with Chr6 polysomy and 19 with neutral VEGFA copy number were assessed by immunohistochemistry for CD68 (marker for M1/M2 macrophages), CD163 (M2 macrophages), programmed death 1(PD-1)- tumor infiltrating and stromal lymphocytes as well as tumoral and stromal PD-1 ligand (PD-L1) expression. CRCs with VEGFA amplification or Chr6 polysomy were associated with decreased M1/M2 macrophages, reduced PD-1-expressing lymphocyte infiltration, as well as reduced stromal expression of PD-L1 at the tumor front. Compared to intermediate-grade CRCs, high-grade CRCs were associated with increased M1/M2 macrophages and increased tumoral expression of PD-L1. Our results suggest that VEGFA amplification or Chr6 polysomy is associated with an altered tumor immune microenvironment.

Clinicopathological Features and Metastatic Pattern of Hepatocellular Carcinoma: An Autopsy Study of 398 Patients.

OBJECTIVES: Analysis of a large local autopsy collective to gather epidemiological and histopathological data on hepatocellular carcinoma (HCC). METHODS: We examined a large dataset of 44,104 autopsies performed at the Institute of Pathology, Basel, Switzerland, including 2 autopsy collectives (1969-1983 and 1988-2012) to gather current data on HCC in the advanced stage. A total of 398 HCC were diagnosed, accounting for around 1% of all autopsies. RESULTS: As expected, most patients developing HCC had advanced stages of liver fibrosis or cirrhosis (F3/F4). However, in the more recent autopsy collective (1988-2012), our data also show an increase of HCC arising in livers without or with only mild to moderate fibrosis (F0-F2). Extrahepatic metastasis was found in 156 of 398 HCC (39.1%), with lung metastasis (74.5%) being the most common, followed by the bones (24.8%) and adrenal glands (19.1%). CONCLUSIONS: Our data therefore seem to suggest that, in the last 2 decades, despite the introduction of new therapeutic modalities for HCC, no significant changes have been observed regarding the metastatic pattern of advanced HCC.

HMGA1 overexpression is associated with a particular subset of human breast carcinomas.

OBJECTIVES: Breast cancer represents the second leading cause of cancer mortality among American women and accounts for more than 40 000 deaths annually. High-mobility group A1 (HMGA1) expression has been implicated in the pathogenesis and progression of human malignant tumours, including breast carcinomas. The aim of this study was to evaluate HMGA1 detection as an indicator for the diagnosis and prognosis of human breast carcinoma. METHODS: HMGA1 expression has been analysed by immunohistochemistry in a large series of breast carcinoma resections (1338) combined on a tissue microarray mainly including the ductal carcinoma variant. The results were then correlated with clinicopathological parameters of patients. RESULTS: HMGA1 overexpression was found in the large majority of breast carcinoma samples and its overexpression positively correlated with HER-2/neu amplification and progesterone receptor, while a negative correlation was found with oestrogen receptor. Conversely, no HMGA1 expression was found in normal breast tissues. CONCLUSIONS: The data reported here indicate that HMGA1 is overexpressed in human breast carcinomas and its levels are associated with a particular endocrine status.

Upregulation of the miR-17-92 cluster and its two paraloga in osteosarcoma - reasons and consequences.

Osteosarcomas (OS) are aggressive bone tumors characterized by complex karyotypes with highly variable structural and numerical chromosomal aberrations. Although several genes and pathways commonly altered in malignant tumors have also been identified in OS, the molecular pathogenesis and driving genetic events eventually leading to tumor development are still poorly understood. The microRNA (miRNA) cluster 17-92 and its two paraloga 106a-363 and 106b-25 are known to have diverse oncogenic properties and have been shown to be constantly upregulated in several established OS cell lines. In this study we analyzed a series of 75 well characterized pretherapeutic OS samples for their expression of cluster-related miRNAs and correlated our findings with clinico-pathological parameters including prognosis, metastases and response to neoadjuvant therapy. Interestingly, higher expression levels of specific miRNAs were significantly associated with an adverse outcome of patients and were also higher in patients with systemic spread. We could furthermore show a direct correlation between the expression of cluster activators (MYC, E2F1-3), inhibitors (TP53), individual miRNAs, and pro-apoptotic targets (FAS, BIM). Our findings therefore underline a critical role of the miR-17-92 cluster and its two paraloga in OS biology with pathogenetic and prognostic impact.

A differential proteomic approach reveals an evolutionary conserved regulation of Nme proteins by Fe65 in C. elegans and mouse.

The function of the APP-Fe65 complex is still not definitively understood. To address this point we studied the phenotype of Fe65 (feh-1) ablation, which results in severe developmental defects in C. elegans, including embryonic and larval arrests. To shed light on the complex phenotype of embryonic arrest, we undertook a systematic approach, aiming at the definition of the altered proteomic profile of feh-1 null worms. We defined a panel of 27 regulated proteins, 16 of which actually participating to embryonic development processes in the nematode. Protein spots corresponding to the products of the F25H2.5 gene, the nematode orthologue of mammalian Nm23/Nme gene family members, were consistently up-regulated in feh-1 -/- embryos. We observed similar up-regulation of Nme1 and Nme2 genes, both at the transcript and the protein levels, in the brain of Fe65 knock-out mice, thus highlighting the occurrence of evolutionary conserved mechanisms of Nme expression in nematodes and mammals.