Piscuoglio, Salvatore

Intratumoral heterogeneity and clonal evolution are pivotal in the progression and metastasis of melanoma. However, when combined with variable tumor cellularity, intratumoral heterogeneity limits the sensitivity and accuracy of uncovering a cancer’s clonal evolution. In this study, we combined fluorescence-activated cell sorting (FACS) with whole-exome sequencing (WES) to investigate the clonal composition and evolutionary patterns in seven melanoma biopsies obtained from three patients, each having both primary site and metastatic samples. We employed a multiparameter ploidy sorting approach to isolate tumor populations based on DNA ploidy and melanoma biomarkers (SOX10 or S100), enabling us to investigate clonal evolution with high resolution. Our approach increased the mean tumor purity from 70% (range 19–88%) in unsorted material to 91% (range 87–96%) post-sorting. Our findings revealed significant inter- and intratumor heterogeneity, with one patient exhibiting two genomically distinct clonal tumor populations within a single primary site biopsy, each giving rise to different metastases. Our findings highlight the critical role of intratumoral heterogeneity and clonal evolution in melanoma, especially when analyzing tumor trajectories. The unique combination of multiparameter FACS and WES provides a powerful method for identifying clonal populations and reconstructing clonal evolution. This study provides valuable insights into the clonal architecture of melanoma and lays the groundwork for future research with larger patient groups.

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.

AbstractHomologous recombination (HR) DNA repair‐deficient (HRD) breast cancers have been shown to be sensitive to DNA repair targeted therapies. Burgeoning evidence suggests that sporadic breast cancers, lacking germline BRCA1/BRCA2 mutations, may also be HRD. We developed a functional ex vivo RAD51‐based test to identify HRD primary breast cancers. An integrated approach examining methylation, gene expression, and whole‐exome sequencing was employed to ascertain the aetiology of HRD. Functional HRD breast cancers displayed genomic features of lack of competent HR, including large‐scale state transitions and specific mutational signatures. Somatic and/or germline genetic alterations resulting in bi‐allelic loss‐of‐function of HR genes underpinned functional HRD in 89% of cases, and were observed in only one of the 15 HR‐proficient samples tested. These findings indicate the importance of a comprehensive genetic assessment of bi‐allelic alterations in the HR pathway to deliver a precision medicine‐based approach to select patients for therapies targeting tumour‐specific DNA repair defects. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract Background: Germline mutations in the BRCA1 and BRCA2 genes lead to hereditary breast cancers that are defective in homologous recombination (HR) repair and sensitive to DNA damaging agents. HR deficiency (HRD) also occurs in sporadic breast cancers, but its incidence and etiology are unclear. Genomic signatures of HRD recently were employed as biomarkers in clinical trials with modest success. We posited that sporadic breast cancers displaying functional HR deficiency would harbor genetic alterations affecting HR DNA repair genes. To test this hypothesis, we applied a functional assay to define lack of competent HR DNA repair and sequenced the exomes of consecutive sporadic breast cancers. Methods: We developed an assay to assess the ability of cancer cells to localize RAD51 into sub nuclear foci in response to ex-vivo irradiation (IR) in fresh sporadic breast cancer tissue specimens from 60 patients. RAD51 focus formation was compared between mock and IR conditions to determine relative fold induction. Twenty-nine tumors with sufficient DNA underwent whole-exome sequencing. Structural genomic signatures of HRD (i.e. large state transitions (LST), telomeric imbalance (NtAI), loss-of-heterozygositiy (LOH)) and a previously reported mutational signature related to BRCA1/2 hereditary breast cancers were assessed. HR deficient tumors were defined as those with both RAD51 foci defects and a genomic signature of HR deficiency (LST>15 or presence of a BRCA mutational signature). Somatic, germ-line, and copy number changes in HR genes were investigated. BRCA1 methylation status was determined. Results: Seventeen of 60 (28%) tumors displayed defective RAD51 recruitment following ex-vivo IR (RAD51-DEF). RAD51-DEF was seen in all breast cancer subtypes , including 7 of 33 (21%) ER+/HER2-, 4 of 14 (29%) HER2+, and 6 of 13 (48%) triple-negative cases. Of the 29 sequenced tumors, 13 (45%) were RAD51-DEF and 16 (55%) were competent for inducing RAD51 foci. LST was elevated in 10 tumors (LST >15) and associated with RAD51-DEF (p=0.02), whereas NtAI (p=0.10) and LOH (p=0.052) did not show a significant association with RAD51-DEF. The BRCA1/2 mutational signature was evident in 4 tumors, all were RAD51-DEF (p=0.03) and 2 were BRCA2 mutated. Nine of 29 (31%) sequenced tumors were determined to have HRD by the RAD51 assay and presence of a genomic scar. Eight of these 9 (88%) cases with HRD had a genetic alteration of both alleles of a bona fide HR gene due to a pathogenic mutation (somatic or germline) coupled with loss of heterozygosity or a homozygous deletion compared to 1 (5%) tumor without HRD (p<0.001). Both alleles of a gene were affected for BRCA2 (n=4), FAAP100 (n=2), CHEK2 (n=1), TP53BP1 (n=1) and BRCA1 (n=1). BRCA1 gene promoter methylation was found not to be significantly associated with HRD. Conclusion: Combined functional and genomic analyses of breast tumors demonstrated that genetic loss of an HR gene may underpin HRD in sporadic breast cancers. Our findings warrant further comprehensive genetic assessment (somatic, germline, and copy number) of HR genes as potential biomarker for HR-directed therapies. Citation Format: Powell SN, Riaz N, Mutter RW, Ng CKY, Delsite R, Piscuoglio S, King TA, Martelotto L, Sakr R, Brogi E, Edelweiss M, Lim R, Higginson D, Weigelt B, Lee W, Reis-Filho JS. A functional assay for homologous recombination (HR) DNA repair and whole exome sequencing reveal that HR-defective sporadic breast cancers are enriched for genetic alterations in DNA repair genes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-03.