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Lineage plasticity in cancer reflects the property of neoplastic cells to adapt to external pressures by switching between different developmental pathways. While some prostate cancers appear “constrained” to a luminal epithelial lineage despite progression on androgen signaling inhibitors, others exhibit a “lineage-promiscuous” phenotype under the pressures of these therapies. However, a clinically applicable marker that quantifies lineage plasticity has yet to be developed. To address this knowledge gap, we applied single-cell genomics and bulk epigenetic profiling to two patient derived xenografts (PDXs) derived before and after chemotherapy from the castration-resistant prostate tumor of a 42-year-old with a PSA 486, Gleason 5+4, cT4N1M1b PCa, who succumbed to his disease within 21 months of diagnosis. We reasoned that the two PDXs, MDA PCa 177-B (AR negative, basal transcriptional profile) and MDA PCa 189-1 (AR positive, luminal transcriptional profile), should share the property of lineage plasticity and serve to define candidate measurable signatures of this property. Targeted and single-cell DNA sequencing showed that the two models share 66.8% of their annotated mutations, 71.5% of copy number variations, and a common ancestor in their phylogeny, confirming their common clonal origin. Single-cell RNA sequencing revealed the presence of a cluster of cells shared by both PDXs that contained a highly mixed program, similar to a “high-plasticity cell state” (HPCS) previously implicated in the emergence and maintenance of cellular heterogeneity in genetically engineered mouse models of lung cancer (Marjanovic et al PMID: 32707077). H3K27ac, H3K27me3 and H3K4me3 chromatin immunoprecipitation followed by sequencing and reduced representation bisulfite sequencing performed in triplicates revealed chromatin profiles shared by both PDX models, and being absent from normal prostate specimens in publicly available datasets. In conclusion, our data supports the hypothesis that the property of lineage plasticity can be reflected in a biomarker signature and used as a measurable metric. Ongoing are evaluation of the candidate signatures in publicly available datasets representative of the spectrum of the disease in patient samples, and assessment of the effects of systemic therapies (including epigenetic modulators) on the property of lineage plasticity in clinical and co-clinical samples. Citation Format: Souzana Logotheti, Haswanth Vundavilli, Eugenia Papadaki, Yuehui Zhao, Marcos Roberto Estecio, Rama Soundararajan, Peter Shepherd, Jiabin Dong, Anh Hoang, Shuai Guo, Nora Navone, Vasiliki Tzelepi, Christopher Logothetis, Yue Lu, Aristidis Vrahatis, Wenyi Wang, Ana Aparicio. Candidate measures of lineage plasticity in aggressive phenotypes of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 142.

428 Background: Bilateral TGCT offers a unique opportunity to elucidate a stem cell versus genetic origin of cancer (Coffey, 2008). Comparison of the epigenetics and genetics of disparate tumors in either synchronous or metachronous bilateral TGCT from the same patients is feasible and may be informative. Methods: We examined the clinical characteristics and natural history of 37 patients with bilateral TGCT. We performed reduced representation bisulfite sequencing (RRBS) of FFPE DNA and whole exon sequencing (WES) of 5 of those patients whose tumor samples were available for the study. Results: We identified 37 patients with bilateral TGCT, who underwent their first orchiectomy between January 1984 and April 2022 and the second between August 1997 and April 2022. Thirty-five (95%) of these patients are still alive. Seven patients had synchronous, while 30 had metachronous bilateral TGCT. There were 13 bilateral seminomas, 14 bilateral nonseminomas, and 10 bilateral seminoma and nonseminoma. For those patients with metachronous bilateral TGCT, the median time between the two TGCT was 59 months (range 6-400). WES revealed a mutation in 189 (

423 Background: There is debate whether a primary seminoma of the anterior mediastinum originates from a stranded germ cell in the midline near the thymus as it migrates from the yolk sac to the gonad1 or from abnormal development of the thymic anlage during embryogenesis.2 We hypothesized that if extragonadal seminoma were to arise from an adult stem cell at an ectopic site rather than from an errant primordial germ cell (PGC), it would display a methylated imprint resembling that of thymic epithelial tumors, such as a thymoma, rather than that of a gonadal seminoma. Methods: We performed reduced representation bisulfite sequencing (RRBS) of FFPE DNA of testicular seminoma, anterior mediastinal seminoma, thymomas, atypical thymomas, and thymic carcinomas. Results: RRBS of testicular seminoma (n=1), anterior mediastinal seminoma (n=1), thymomas (n=2), atypical thymomas (n=2), and thymic carcinomas (n=2) showed that the methylated genetic imprint of an extragonadal seminoma clustered more closely to that of an atypical thymoma than that of testicular seminoma. Conclusions: Our preliminary RRBS results suggest that extragonadal seminoma may have a thymic rather than PGC origin. We plan to confirm these results by evaluating certain PGC-specific genetic biomarkers and epigenetic signatures in additional cases. References: 1. Dixon FJ, Moore RA. Testicular tumors: a clinicopathological study. Cancer 1953;6:427. 2. Schlumberger HG. Teratoma of the anterior mediastinum in the group of military age: a study of sixteen cases, and a review of theories of genesis. Arch Pathol 1946;41:398.

SUMMARYHow anti-cancer immunity shapes early carcinogenesis of lung adenocarcinoma (ADC) is unknown. We characterized immune contexture of invasive lung ADC and its precursors by transcriptomic immune profiling, T cell receptor (TCR) sequencing and multiplex immunofluorescence. Our results demonstrated that anti-tumor immunity evolved as a continuum from lung preneoplasia, to preinvasive ADC, minimally-invasive ADC and frankly invasive lung ADC with a gradually less effective and more intensely regulated immune response including down-regulation of immune-activation pathways, up-regulation of immunosuppressive pathways, higher infiltration of CD4+ T cells, lower infiltration of CD8+ T cells, decreased T cell clonality, and lower frequencies of top T cell clones in later stages. Driver mutations, HLA loss, chromosomal copy number aberrations and DNA methylation changes may collectively impinge host immune responses and facilitate immune evasion as a potential mechanism underlying outgrowth of the most fit subclones in preneoplasia into dominant clones in invasive ADC.SIGNIFICANCEThere has been a drastic increase in the detection of lung nodules, many of which are lung ADC precursors. The management of these lung nodules is controversial. We discovered that immune activation and evasion have started at preneoplastic stage and lung ADC precursors may exhibit an overall better-preserved anti-tumor immune contexture suggesting therapeutic strategies reprograming the immune microenvironment in patients with lung ADC precursors prior to further immunosuppression in invasive lung cancers may be beneficial. These findings have served as the critical scientific rationale for our immunoprevention clinical trial IMPRINT-Lung (NCT03634241) recruiting individuals diagnosed with lung nodules at high risk developing invasive lung cancers.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription–quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options The work in GAC’s laboratory was partly supported by NIH/National Center for Advancing Translational Sciences grant UH3TR00943-01, the NIH/National Cancer Institute (NCI) grant 1 R01 CA182905-01, U54 grant UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, Team Department of Defense grant CA160445P1, a Ladies Leukemia League grant, a Chronic Lymphocytic Leukemia Moonshot Flagship project, a Sister Institution Network Fund 2017 grant, and the Estate of C. G. Johnson, Jr. EFM was supported in part by award number P50 CA140388 from the NCI and by the NIH Clinical Research Loan Repayment Program. AMS was supported by Fundação para a Ciência e a Tecnologia through fellowship SFRH/BD/85968/2012. ZJZ’s work was supported by grants from the MPN Foundation and the Oklahoma Center for the Advancement of Science and Technology. DG, MPD, and IBN were supported in part by a Programul Opera¿ional Competitivitate grant nr35/01.09.2016, ID 37_796, titled “Clinical and economical impact of personalized targeted anti-mi-croRNA therapies in reconverting lung cancer chemoresistance” (CANTEMIR). NSDA was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, BEPE 2016/09349-4.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.