Thomas Lorber

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.

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

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.

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine.

Introduction The use of patients’ own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs. Methods MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists. Results We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs. Conclusions We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

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 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.

Abstract 3424: Deciphering the genomic heterogeneity and evolution in malignant melanoma by genomic profiling of clonal tumor populations

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background With the recent improvements in genomic analysis tools, research groups all over the world are intensively deep-sequencing different tumor entities, including malignant melanomas. However, most of these studies are using only a single biopsy per patient, providing only limited insights into genomic tumor evolution. In this study, we performed genomic profiling of sorted clonal tumor populations from several specimens per patient in order to infer genomic heterogeneity in malignant melanomas and genomic evolution during tumor progression. Methods A multi-parameter sorting approach was used to flow-sort multiple biopsies from individual melanoma patients. Therefore nuclei were extracted from frozen and FFPE tissues and sorted according to DNA ploidy. SOX10 was used as an additional melanoma parameter to ensure sorting of pure tumor populations. Resulting clonal tumor populations were genomically characterized by usage of high resolution aCGH and deep-sequencing with NGS technologies. Results Array CGH and NGS performed on flow-sorted clonal tumor populations (DNA and SOX10) revealed the clonal relationship between multiple tumor biopsies from individual patients. The tumor marker SOX10 allowed us to uncover and sort pure tumor populations, excluding the risk of contribution of non-neoplastic cells in downstream analysis with aCGH and NGS. Taken together the information of ploidy, aCGH and NGS data, we were able to detect chromosome imbalances and allelic frequencies from these populations. Our approach allows us to track the evolution of clonal populations across time and organs and in the context of therapeutic interventions. Conclusions Human malignant melanomas are composed of different clonal populations with population-specific genomic aberrations and mutations. The use of SOX10 allowed unraveling of pure tumor populations within the diploid peaks, which would have been obscured by the use of DNA content alone. NGS and aCGH helped to characterize these sorted clonal tumor populations. Further bioinformatic analyses of these sorted clonal tumor populations are fundamental for the understanding of the clonal relationship and genomic heterogeneity and their potential impact on metastasis and therapy response in malignant melanoma. Citation Format: Thomas Lorber, Tanja Dietsche, Valeria Perrina, Michael Barret, Kathrin Glatz, Christian Ruiz, Lukas Bubendorf. Deciphering the genomic heterogeneity and evolution in malignant melanoma by genomic profiling of clonal tumor populations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3424. doi:10.1158/1538-7445.AM2014-3424

Abstract 3167: Deciphering the genomic heterogeneity in malignant melanoma by genomic profiling of clonal tumor populations.

1) Background Melanoma is a heterogeneous disease and even though consistent genetic patterns among different subtypes have been revealed, clinical trials have failed to significantly improve survival. However, most of the genomic studies do not take into consideration the underlying genomic heterogeneity. In this study, we used genomic profiling of sorted clonal tumor populations in order to investigate the genomic heterogeneity and the clonal evolution in malignant melanoma. 2) Methods Multiple biopsies from melanoma patients were subjected to flow-sorting based on DNA content. The tumor specific marker SOX10 was used as an additional parameter to exclude contribution of normal tissue. Resulting clonal tumor populations were genomically characterized by usage of high resolution aCGH. A TMA was constructed for validation of the findings. 3) Results DNA content based flow sorting of small human melanoma biopsies was successfully implemented and revealed the co-existence of distinct clonal populations (diploid as well as aneuploid) within these samples. The use of the additional marker SOX10 allowed us to uncover and sort pure diploid tumor populations from diploid fractions admixed with normal cells. These findings were confirmed by aCGH. 4) Conclusions Human malignant melanoma is composed of different clonal populations with population-specific genomic aberrations. The use of SOX10 allowed unraveling of pure tumor populations within the diploid peaks, which would have been obscured by the use of DNA content alone. Further genomic analyses of these sorted clonal tumor populations are fundamental for the understanding of the genomic heterogeneity and its potential impact on metastasis and therapy response in malignant melanoma. Citation Format: Thomas Lorber, Tanja Dietsche, Joel Gsponer, Alexander Rufle, Michael Barret, Lukas Sommer, Katharina Glatz, Christian Ruiz, Lukas Bubendorf. Deciphering the genomic heterogeneity in malignant melanoma by genomic profiling of clonal tumor populations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3167. doi:10.1158/1538-7445.AM2013-3167