Perrina, Valeria

Abstract Background:All carcinomas contain a variable proportion of benign stromal and immune cells, limiting the sensitivity in the identification of somatic genetic alterations. The average tumor purity of squamous cell carcinomas (SCC) of the lung is only around 50%. Low tumor content in tumor samples represents a challenge in studying intratumoral genomic heterogeneity in cancer research. Here, we applied flow-sorting to enrich for tumor cell nuclei to investigate the clonal relationship of primary SCC and matched metastases. Methods:Tumor tissues from 16 patients with primary SCC of the lung and matched metastases were used. We implemented a flow-sorting based approach to enrich for tumor nuclei as followed: after extraction of nuclei from snap-frozen or FFPE tissue, they were flow-sorted by DNA content (ploidy) and cytokeratin expression of the adherent cytoplasm, using a pan-cytokeratin (pCK) antibody. Isolated diploid and aneuploid pCK-positive tumor cell populations were subjected to whole exome sequencing (WES). DNA from diploid pCK-negative populations was used as germline control. Mutational profile and copy number aberrations (CNA) were determined to infer the clonal relationship and evolution between the primary tumors and their metastases. Results:Our flow-sorting based approach was able to enrich tumor content from 20%-50% to more than 80%-90% and to distinguish between aneuploid and diploid tumor cell populations from bulk tumor tissues. It enabled the identification of somatic mutations and CNA in both, aneuploid and diploid tumor cell populations, including potential subclonal driver mutations in ARID1A and KDM6A at low allelic frequencies. Shared and private mutations were observed in matched longitudinal tumor samples of individual patients and in synchronous distant metastases. Ploidy did not change significantly between primary tumors and relapse or distant metastases. Conclusion:We present a flow-sorting based method to enrich for tumor cell nuclei to facilitate genomic analysis, which also enabled separate analysis of aneuploid and diploid tumor populations. Our results show that the enrichment approach can be used to decode the clonal evolutionary relationship between primary tumors and their matched metastases, even in samples with low tumor cell content. Citation Format: Arthur Krause, Maria R. De Filippo, Thomas Lorber, Tanja Dietsche, Valeria Perrina, Christian Ruiz, Michael T. Barrett, Salvatore Piscuoglio, Charlotte K. Ng, Lukas Bubendorf. Enriching tumor purity using a unique flow-sorting approach to elucidate clonal evolution in matched samples of squamous cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4699.

AbstractVariable 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.

Abstract Background: Genomic intratumoral heterogeneity in cancers is a driving force for progression or recurrence. Availability of pure tumor cell material is critical for studying clonal evolution. In general, tumors are highly mixed with benign cells with a mean tumor cell purity of only 49%1 shown in lung squamous cell carcinoma (LSCC). Using a unique flow-sorting based approach enables us to overcome this limitation by highly enriching for tumor cells. Here, we aimed at precisely identifying different tumor populations in primary LSCC and matched distant metastases from 18 patients. Methods: Nuclei from snap-frozen and FFPE tumor specimens were extracted and subjected to flow-sorting using DNA content/ploidy as a parameter. Pan-cytokeratin (pCK) was used to differentiate diploid tumor cell populations from benign diploid stromal cells. DNA has been extracted from these individual tumor cell populations and the mutational profile and copy number status are currently being analyzed using whole exome seqeuencing (WES). Results: We successfully enriched for tumor cells with a purity of higher than 95% using the multi parameter flow-sorting approach. Besides the aneuploid tumors with higher ploidies, we discovered near-diploid tumor populations after enrichment with the pCK antibody. The pCK negative fraction consisting of stromal cells can serve as a germ line control in further downstream analysis as WES. Conclusion: The present study illustrates a unique method that allows high enrichment for tumor cells. Avoiding the dilution of DNA by benign stromal cells, together with the purity of aneuploid tumor cells, the precision of genomic analysis highly increases. At the same time, the diploid and pCK negative stromal cells can serve as a germ line control. Comprehensive genomic profiling of flow-sorted aneuploid and pCK positive diploid tumor populations represent a powerful technique to study intratumoral heterogeneity. References: 1. Zack, T. I. Pan-cancer patterns of somatic copy number alteration. Nature Genetics. 45, 10 (2013). Citation Format: Arthur Krause, Thomas Lorber, Valeria Perrina, Michael T. Barrett, Christian Ruiz, Lukas Bubendorf. Enrichment and isolation of tumor cells using a unique flow-sorting approach to decrypt intratumoral heterogeneity in matched samples of squamous cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2184.

AbstractUnderstanding 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.

Abstract 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 (<277x) contributes to an overestimation of genomic heterogeneity (Zhang et al., Science 2014). Integrational analysis of ploidy, chromosomal aberrations and mutations in 409 cancer genes allowed to draw the evolutionary picture of each patient's disease. MPP appears as a promising tool for identification of genomic vulnerabilities that could be exploited for tailored treatment. 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 Background: Genomic tumor heterogeneity is one reason for recurrence and resistance to cancer therapy. It is still not known what leads to the relapse or the emergence of metastasis in individual patients. To contribute to answer this question, we investigated the evolutionary patterns in squamous cell carcinoma of the lung (SCC) in 10 patients with multiple matched tumors (primary vs recurrence/metastasis). Methods: To identify the genomic profile, frozen tissues from SCC patients were subjected to multiparameter ploidy profiling (MPP). Tumor populations were sorted based on ploidy (DNA-content using DAPI) and p40 was used to control for diploid tumor populations. Subsequent genomic profiling by array comparative genomic hybridization (aCGH) and targeted massive parallel sequencing (Ion Torrent Comprehensive Cancer Panel with an all-exon coverage of 409 cancer genes) was applied. Results: The usage of MPP enabled the determination of the clonal composition of the SCC and define genomic characteristics. Due to flow sorting the tumor populations were effectively isolated and existence of distinct clonal populations was indicated. Aneuploid and diploid tumors were successfully revealed using the additional marker p40. Therefore, the absence of non-malignant cells increased the precision in the genomic analyses Conclusion: The study uncovered the composition of human SCC in the sense of different clonal populations, which harbor private genomic aberrations and mutations confirmed by aCGH and NGS. Even if our data is still preliminary we provide evidence that applying MPP increases the precision of aCGH and sequencing analysis. Genomic analyses of sorted pure tumor population allows to reconstruct the clonal evolution and facilitates the understanding of tumor heterogeneity and their evolutionary pattern, which could have potentially an impact on therapeutic response or metastasis development. Citation Format: Arthur Krause, Thomas Lorber, Valeria Perrina, Tanja Dietsche, Michael Barrett, Christian Ruiz, Lukas Bubendorf. The analysis of the genomic evolution of squamous cell carcinoma of the lung. [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 155.

Abstract Background Genomic intratumor heterogeneity is an increasingly recognized phenomenon in various solid tumors and is now extensively being investigated by next-generation sequencing (NGS) approaches. However, most methods are not designed to resolve the complexity of mixed cell populations. Therefore important information may be lost, eventually leading to assumptions that may not reflect the true nature of the disease. We have therefore developed the method of Multiparameter Ploidy Profiling (MPP). We applied this technology on tumor specimens from malignant melanoma and from non-small cell lung cancer (NSCLC). Methods MPP involves isolation of nuclei from tissues, multiparameter flow-sorting of tumor populations by DNA content and specific tumor markers, and profiling genomes using high resolution array comparative genomic hybridization (aCGH) and NGS. As some tumors are diploid by flow cytometry, specific lineage markers, e.g. SOX10 (melanoma) and TTF1 (NSCLC) were used to separate these from normal cells. Fluorescence-in-situ hybridization (FISH) and immunohistochemistry (IHC) were performed on sorted nuclei and on whole tissue sections to validate results. Results MPP allowed us to sort different tumor populations within a single tumor specimen, while excluding non-neoplastic contamination in downstream analysis with aCGH and NGS. We separated three different tumor populations from a single primary NSCLC with a TTF1 multiparameter sort. We detected significant differences in copy numbers, e.g. a CDKN2A homozygous deletion only present in the diploid tumor population as well as differences in allelic frequencies of gene mutations (e.g. EGFR exon 20). Further sequencing of the diploid TTF1 negative population proved to be concordant to genomic DNA obtained from a tumor free lymph node. Finally, we used the population-specific genomic data in order to infer the clonal relationship of all tumor populations and to postulate the evolution of these tumor populations within single tumor specimens. Conclusions The power of MPP consists of sorting pure tumor populations and of deciphering the level of intratumor heterogeneity based on ploidy. Most importantly MPP enables the separation of diploid tumor populations from diploid, benign stromal cells that can serve as reference for genomic profiling studies, where germline DNA is otherwise not available. To our knowledge, diploid tumor populations were for first time purifyed from normal cells in a multiparameter sort and genomically characterized. We also demonstrated that the analysis of diploid tumor populations within a single biopsy is crucial for the understanding of the clonal composition and evolution of a given tumor. MPP on multiple biopsies from individual patients is necessary to deeply investigate the clonal evolution and the behavior of a diploid tumor population and their potential impact on metastasis and therapy response. Citation Format: Thomas Lorber, Sabrina Rau, Valeria Perrina, Michael Barrett, Christian Ruiz, Lukas Bubendorf. Multiparameter ploidy profiling: a powerful tool to investigate the genomics of diploid tumor populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4924. doi:10.1158/1538-7445.AM2015-4924