Valeria Perrina

Distinct genetic evolution patterns of relapsing diffuse large B-cell lymphoma revealed by genome-wide copy number aberration and targeted sequencing analysis.

Recurrences of diffuse large B-cell lymphomas (DLBCL) result in significant morbidity and mortality, but their underlying genetic and biological mechanisms are unclear. Clonal relationship in DLBCL relapses so far is mostly addressed by the investigation of immunoglobulin (IG) rearrangements, therefore, lacking deeper insights into genome-wide lymphoma evolution. We studied mutations and copy number aberrations in 20 paired relapsing and 20 non-relapsing DLBCL cases aiming to test the clonal relationship between primaries and relapses to track tumors’ genetic evolution and to investigate the genetic background of DLBCL recurrence. Three clonally unrelated DLBCL relapses were identified (15%). Also, two distinct patterns of genetic evolution in clonally related relapses were detected as follows: (1) early-divergent/branching evolution from a common progenitor in 6 patients (30%), and (2) late-divergent/linear progression of relapses in 11 patients (65%). Analysis of recurrent genetic events identified potential early drivers of lymphomagenesis (KMT2D, MYD88, CD79B and PIM1). The most frequent relapse-specific events were additional mutations in KMT2D and alterations of MEF2B. SOCS1 mutations were exclusive to non-relapsing DLBCL, whereas primaries of relapsing DLBCL more commonly displayed gains of 10p15.3–p12.1 containing the potential oncogenes PRKCQ, GATA3, MLLT10 and ABI1. Altogether, our study expands the knowledge on clonal relationship, genetic evolution and mutational basis of DLBCL relapses.

Donor-derived, metastatic urothelial cancer after kidney transplantation associated with a potentially oncogenic BK polyomavirus: Oncogenic BK virus and donor kidney-derived urothelial cancer

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

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.

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.

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.

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.This paper describes the enrichment of rare neoplastic Hodgkin and Reed-Sternberg (HRSC) cells of classical Hodgkin lymphoma (cHL) from the archival formalin-fixed and paraffin-embedded tissues. This technique allows enrichment of HRSC from 1-5% in the initial cHL tissue to 40-80% in sorted populations, which enables robust genetic analysis of cHL by targeted high throughput sequencing and array-CGH.

Exploring the spatiotemporal genetic heterogeneity in metastatic lung adenocarcinoma using a nuclei flow-sorting approach: "Genomics of flow-sorted primary-metastatic pairs in lung cancer"

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

Delineation of human prostate cancer evolution identifies chromothripsis as a polyclonal event and FKBP4 as a potential driver of castration resistance: Chromothripsis and FKBP4 in advanced prostate cancer

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

Abstract 3178: Metastatic urothelial cancer 10 years after transplanting a filial kidney: genomically investigating the perpetrator

BACKGROUND Polyomavirus (BKV) infection has been associated with development of urothelial carcinoma (UC). We report on a patient with conventional UC of the bladder (Ta, G3) 8 years after receiving a kidney graft from a 4 year old male donor. After a recurrence (T1, G3) 9 years after transplantation (NTx), he was diagnosed with a SV-40 positive metastatic micropapillary and muscle-invasive UC 10 years after NTx with involvement of the graft kidney pelvis (pT3), the bladder (pT3) and a pelvic lymphnode metastasis. The aim of this study was to investigate the genealogy and clonal relationship between these cancer locations. METHODS Cancer cell nuclei derived from formalin fixed tumor specimens were processed and sorted based on DNA content with a BD Influx FACS. DNA from sorted aneuploid populations was analyzed by whole genome high resolution CGH microarrays (aCGH). In addition, short tandem repeat (STR) analyses were done with the PowerPlex® ESI Kit commercial testing kit. Each run was performed on an Applied Biosystems Genetic Analyzer 3500 and analyzed with the GeneMapper ID-X Software. Integration of PV-genom was investigated by RT-PCR. The IonTorrent platform by LifeTech (PGM, IonAmpliseq Cancer Hotspot Panel v2) was used for next-generation-sequencing (NGS). RESULTS Array CGH clarified that the primary two non-muscle invasive bladder tumors differed completely from each other and from the subsequent cancer tissues. The tumor manifestations 10 years after NTx shared the same genomic profile, except for a private amplification at 6p12.3 and a private deletion at Xp22.33 - Xp22.11, both in the bladder tumor. STR identified all three tumors diagnosed 10 years after NTx to originate from the allograft donor. NGS identified two shared mutations of unknown relevance (KDR / InDel chr4:55962545; Akt1 / c.138C>A) in the 10 year after NTx UC sites. No known somatic mutation was found. RT-PCR identified the PV to be integrated in the human cancer genome. Further studies are currently ongoing to define the exact integration sites of BKV and the potentially influenced human genes. CONCLUSIONS This case is an impressive example of a, donor-derived, metastatic micropapillary UC developed under immuno-suppression and subsequent BKV-reactivation in a presumably healthy transplanted kidney of a four years old donor. Citation Format: David C. Muller, Maarit Raemoe, Christian Wetterauer, Valeria Perrina, Luca Quagliata, Tatjana Vlajnic, Christian Ruiz, Beate Balitzki, Hans H. Hirsch, Rainer Grobholz, Lukas Bubendorf, Cyrill A. Rentsch. Metastatic urothelial cancer 10 years after transplanting a filial kidney: genomically investigating the perpetrator. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3178.

Abstract 158: Clonal evolution and genomic tumor heterogeneity in non-small cell lung cancer deciphered by multiparameter ploidy profiling

BACKGROUND Genomic intra- and intertumor heterogeneity is one main reason for relapse and resistance to therapy. There is a shortage of studies characterizing the level of genomic intertumoral heterogeneity of known cancer genes in multiple longitudinal biopsies of individual patients. Additionally, a workflow to deconvulate the intermixture of tumor and stromal components is missing. To overcome these limitations and to decipher the genomic heterogeneity and clonal evolution, we developed a method and applied it on matched (primary-recurrence/metastasis) non-squamous, non-small cell lung cancers (NSCLC). METHODS Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin and paraffin embedded (FFPE) tissues, followed by multiparamter flow sorting. Sorted populations were subjected to genomic profiling by high resolution array comparative genomic hybridization (aCGH) and massively parallel sequencing (MPS). DAPI allowed to seperate populations by ploidy and anti-TTF1 antibodies was used to control for tumor nuclei. Array-CGH, combined with ploidy, was used to retrieve genome wide copy numbers. The Comprehensive Cancer Panel that covers all-exons of 409 cancer genes was applied on all sorted tumor and stromal populations to detect somatic mutations and their variant allelic frequency (VAF). RESULTS MPP was successfully applied to 44 frozen or FFPE tissue specimens from 19 patients. Array-CGH and MPS of TTF1-negative, normal cells were concordant to germline controls. Sequencing revealed that 50% of mutations are shared between primary tumors and metastases. Except for one patient, mutations with VAF>0.3 are shared between primary and metastasis. The variant allelic frequency was significantly higher in shared mutations than mutations that were private to one tumor. Besides common activating mutations in EGFR and KRAS we found biallelic inactivation in tumor suppressor genes like KEAP1, NF1, STK11 and TP53. Two clonal evolutionary patterns were found: 1) early and 2) late divergence. Matched tumors without any shared mutations were classified as unrelated primary tumors. CONCLUSION The power of MPP is to increase the precision of downstream analysis, due to the sorting of pure populations of tumor cells. It permitted to infer the clonal evolution of tumor populations with unprecedented confidence. The low level of genomic heterogeneity of mutations with VAF>0.3 in this study is in line with recent data from Zhang et al., who showed that the poor precision of low depth sequencing ( Citation Format: Thomas Lorber, Tanja Dietsche, Valeria Perrina, Darius Juskevicius, Arthur Krause, David Mueller, Michael Barrett, Christian Ruiz, Lukas Bubendorf. Clonal evolution and genomic tumor heterogeneity in non-small cell lung cancer deciphered by multiparameter ploidy profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 158.