Barrett, Michael T.

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.

3101 Background: PDO is a promising translational tool that recapitulates the biology and drug response of donor cancer patient. However, an unmet need is to have PDO drug-screening data available for treatment decision making in clinic. We conducted a pilot study to determine whether PDO testing results will be available at critical treatment decision points in metastatic GI cancer patients. Methods: Metastatic GI cancer patients undergoing core-needle biopsy were eligible. Tumor cells isolated from ≤4 fresh biopsy tissues were grown in a Matrigel-based culture. PDO response to anti-cancer drugs were evaluated; and when available, correlated with donors’ clinical response to the same agent(s). PDO response was defined as IC50 < 0.1 × published Cmax of the drug clinically; stable as IC50 between 0.1 to 10 × Cmax. Radiographic response was per RECIST criteria. Results: We enrolled 27 refractory metastatic GI cancer patients (9 colorectal [CRC], 9 pancreas, and 9 biliary tract). Median lines of therapy were 4, 2, and 2; the success rate of organoid establishment was 89%, 44%, and 55%, respectively. The median time from biopsy to availability of drug-testing data was 64 days (range: 24 to 93 days). The median time from biopsy to next CT re-staging in donors was 64 days. The established PDOs shared histological and genomic features with donor clinical tissue. PDO and clinical responses to the same agent(s) were correlated in 2 CRC donors including (1) BRAFV600E-mutated PDO responded to vemurafenib + panitumumab, as did the donor who had partial response (PDO drug-testing data were available 55 days post-biopsy, 23 days prior to restaging scan); (2) KRAS/FGF-dual amplified PDO had stable disease status to regorafenib, as did the biopsied lesion from the donor (73 days post-biopsy, 5 days post-scan). Conclusions: We showed the feasibility of completing PDO drug sensitivity testing in metastatic GI cancer patients within a short time that could impact clinical decision making, particularly in CRC. PDO drug response showed correlation with clinical response. With further refinement, PDO can be a powerful tool for personalizing cancer therapy in metastatic GI cancer patients.

Diffuse large B‐cell lymphoma (DLBCL) is up to 17‐fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (−) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(−) germinal center B‐cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression ( CCNA2 , CCNB1 , CDC25A , E2F1 ), DNA replication ( MCM2 , MCM4 , MCM7 ) and DNA damage repair, including eight Fanconi anemia genes ( FANCA , FANCD1/BRCA2 , FANCE , FANCG , FANCR/RAD51 , FANCS/BRCA1 , FANCT/UBE2T , FANCV/MAD2L2 ), were significantly increased in HIV(+) GCB‐DLBCL tumors compared to HIV(−) tumors. In contrast, genes associated with cell cycle inhibition ( CDKN1A , CDKN1B ) as well as apoptosis regulating BCL2 family members ( BCL2 , BAX , BIM , BMF , PUMA ) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB‐DLBCL tumors have fewer copy number variations than their HIV(−) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB‐DLBCL is a distinct molecular malignancy from HIV(−) GCB‐DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.

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.