Precision Medicine Approaches to Hormone-Driven Cancer

Cancers of the breast and prostate are primarily driven by sex hormone signaling which has been targeted clinically with considerable success. The genomic and transcriptomic landscape of these tumors has been thoroughly elucidated and major advances have been made in non-invasive techniques, however, the full potential of these advances is yet to be realized in the clinic. Two areas of our focus, with such opportunities to improve patient outcomes in hormone-driven cancers are: 1) Non-invasive tracking of metastatic breast cancer (mBC) evolution for precision medicine; 2) Non-invasive early detection of prostate cancer (PCa). mBC is an ultimately treatment-resistant, lethal disease characterized by intra-patient molecular heterogeneity including genomic alterations driving resistance to therapy directed at estrogen receptor (ER; ESR1) signaling (endocrine therapy). Tumor biopsies are not routinely available in this setting. We sought to determine whether circulating tumor cells (CTCs) recapitulate the genomic landscape of bulk tumor tissue. In project 1, we isolated and genomically profiled individual CTCs from 12 patients with mBC who had concurrent whole exome sequencing of their metastatic biopsy bulk tissue. In 76 individual and pooled informative CTCs, we observed 85% concordance in at least one driver somatic mutation/copy number alteration (CNA) between CTCs and matched tissue metastases, with CTC profiling identifying diverse intra- and inter-patient molecular mechanisms. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32) also present in tissue, or harbored a novel ESR1 p.A569S mutation not observed in tissue (n = 1/32), which was demonstrated to be modestly activating in vitro. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived CTCs. Despite advances in biomarker development, early detection of aggressive PCa remains challenging. We previously developed a clinical-grade laboratory-developed test—MiProstate Score (MiPS)—for individualized aggressive PCa risk prediction. MiPS combines serum PSA with transcription-mediated amplification (TMA)-quantified expression of the gene-fusion TMPRSS2:ERG and the lncRNA PCA3 from whole urine obtained after a digital rectal exam (DRE). To improve MiPS, in our second project, we describe the pre-clinical development and validation of a post-DRE whole urine targeted RNA NGS assay (NGS-MiPS) assessing ~90 PCa candidate transcriptomic biomarkers, including: TMPRSS2:ERG.T1E4 and PCA3, additional common PCa gene fusion isoforms, mRNAs, lncRNAs, and expressed mutations. NGS-MiPS showed high analytic validity and was able to detect expressed germline risk HOXB13 and somatic driver SPOP mutations. In an extreme design cohort (benign or Grade Group (GG) 1 vs. GG 3-5 cancer on biopsy) NGS-MiPS showed expected differences in the levels of TMPRSS2:ERG.T1E4, PCA3, and additional biomarkers, between benign/GG 1 vs. GG 3-5 PCa. A machine learning approach trained on a subset of the extreme design cohort (n=73) generated a 15-transcript model that outperformed derived MiPS and serum PSA models in predicting biopsy outcome in two validation cohorts: 1. A held-out set from the extreme design cohort (n=36); and 2. A separate PCa active surveillance cohort (n=45). These results support the potential utility and continued development of our novel urine-based targeted RNA NGS assay to improve aggressive PCa early detection. Leveraging recent technological advances—CTC isolation, NGS, liquid biopsy techniques—as well as knowledge of the genomic landscape of hormone-driven cancers, we demonstrate feasibility of non-invasive precision medicine in mBC and PCa with potential clinical utility.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155106/1/acani_1.pd

Modulation Of Anti-Tumor Immune Response By Tgf-Β-Inducible Early Gene 1 (tieg1)

Cancer immunotherapy has had limited clinical efficacy partly because regulatory T cells (Treg) suppress the immune response to tumor-associated antigens. Inducible regulatory T cells (iTreg), which are converted from naïve CD4 T cells by TGF-β, an abundant cytokine in the tumor microenvironment, may contribute to this immune suppression. Induction of Foxp3 by TGF-β is mediated by the transcription factor TIEG1 and abrogation of this protein prevents Foxp3 expression. We are testing the hypothesis that blockade of TIEG1 to prevent iTreg conversion will enhance immune response in DNA vaccination to the tumor associated antigen Her-2. Wild type and TIEG1 knockout mice in C57BL/6 background were immunized with three different antigens, then immune responses were compared. In TIEG1-/- mice, immunization with allogeneic spleen cells resulted in a stronger T cell response with comparable levels of antibody. Similarly, Her-2-expressing irradiated tumor cells induced a stronger anti-Her-2 T cell response in TIEG1-/- mice, without a significant difference in antibody levels. DNA vaccine encoding human Her-2 also induced a stronger T cell response but with reduced IgG antibody titers specifically in IgG1 and deficient switching to IgG2c. B cells in TIEG1-/- mice had adequate counts in vivo and they proliferated normally in vitro in response to PMA + Ionomycin stimulation. Foxp3+ cells had adequate numbers in TIEG1-/- mice whereas Myeloid Derived Suppressor Cells had stronger proliferation to PMA + Ionomycin stimulation. Initially tumors grew more slowly in TIEG1-/- mice but the difference disappeared later. A new sub strain of TIEG1-/- mice was derived that has similar T cell response but more severely impaired antibody response. Together, these data suggest an amplified T (but not B) cell response in TIEG1-/- mice, indicating pleiotropic immune modulating effects of TIEG1

Comprehensive serial molecular profiling of an “N of 1” exceptional non-responder with metastatic prostate cancer progressing to small cell carcinoma on treatment

Abstract Importance Small cell carcinoma/neuroendocrine prostate cancer (NePC) is a lethal, poorly understood prostate cancer (PCa) subtype. Controversy exists about the origin of NePC in this setting. Objective To molecularly profile archived biopsy specimens from a case of early-onset PCa that rapidly progressed to NePC to identify drivers of the aggressive course and mechanisms of NePC origin and progression. Design, setting, and participants A 47-year-old patient presented with metastatic prostatic adenocarcinoma (Gleason score 9). After a 6-month response to androgen deprivation therapy, the patient developed jaundice and liver biopsy revealed exclusively NePC. Targeted next generation sequencing (NGS) from formalin-fixed paraffin-embedded (FFPE)-isolated DNA was performed from the diagnostic prostate biopsy and the liver biopsy at progression. Intervention Androgen deprivation therapy for adenocarcinoma followed by multiagent chemotherapy for NePC. Main outcomes and measures Identification of the mutational landscape in primary adenocarcinoma and NePC liver metastasis. Whether the NePC arose independently or was derived from the primary adenocarcinoma was considered based on mutational profiles. Results A deleterious somatic SMAD4 L535fs variant was present in both prostate and liver specimens; however, a TP53 R282W mutation was exclusively enriched in the liver specimen. Copy number analysis identified concordant, low-level alterations in both specimens, with focal MYCL amplification and homozygous PTEN, RB1, and MAP2K4 losses identified exclusively in the NePC specimen. Integration with published genomic profiles identified MYCL as a recurrently amplified in NePC. Conclusions and relevance NGS of routine biopsy samples from an exceptional non-responder identified SMAD4 as a driver of the aggressive course and supports derivation of NePC from primary adenocarcinoma (transdifferentiation).http://deepblue.lib.umich.edu/bitstream/2027.42/113670/1/13045_2015_Article_204.pd

Identification of actionable targets for breast cancer intervention using a diversity outbred mouse model

HER2-targeted therapy has improved breast cancer survival, but treatment resistance and disease prevention remain major challenges. Genes that enable HER2/Neu oncogenesis are the next intervention targets. A bioinformatics discovery platform of HER2/Neu-expressing Diversity Outbred (DO) F1 Mice was established to identify cancer-enabling genes. Quantitative Trait Loci (QTL) associated with onset ages and growth rates of spontaneous mammary tumors were sought. Twenty-six genes in 3 QTL contain sequence variations unique to the genetic backgrounds that are linked to aggressive tumors and 21 genes are associated with human breast cancer survival. Concurrent identification of TSC22D3, a transcription factor, and its target gene LILRB4, a myeloid cell checkpoint receptor, suggests an immune axis for regulation, or intervention, of disease. We also investigated TIEG1 gene that impedes tumor immunity but suppresses tumor growth. Although not an actionable target, TIEG1 study revealed genetic regulation of tumor progression, forming the basis of the genetics-based discovery platform

Serial monitoring of genomic alterations in circulating tumor cells of ER-positive/HER2-negative advanced breast cancer: feasibility of precision oncology biomarker detection.

Nearly all estrogen receptor (ER)-positive (POS) metastatic breast cancers become refractory to endocrine (ET) and other therapies, leading to lethal disease presumably due to evolving genomic alterations. Timely monitoring of the molecular events associated with response/progression by serial tissue biopsies is logistically difficult. Use of liquid biopsies, including circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), might provide highly informative, yet easily obtainable, evidence for better precision oncology care. Although ctDNA profiling has been well investigated, the CTC precision oncology genomic landscape and the advantages it may offer over ctDNA in ER-POS breast cancer remain largely unexplored. Whole-blood (WB) specimens were collected at serial time points from patients with advanced ER-POS/HER2-negative (NEG) advanced breast cancer in a phase I trial of AZD9496, an oral selective ER degrader (SERD) ET. Individual CTC were isolated from WB using tandem CellSearch® /DEPArray™ technologies and genomically profiled by targeted single-cell DNA next-generation sequencing (scNGS). High-quality CTC (n = 123) from 12 patients profiled by scNGS showed 100% concordance with ctDNA detection of driver estrogen receptor α (ESR1) mutations. We developed a novel CTC-based framework for precision medicine actionability reporting (MI-CTCseq) that incorporates novel features, such as clonal predominance and zygosity of targetable alterations, both unambiguously identifiable in CTC compared to ctDNA. Thus, we nominated opportunities for targeted therapies in 73% of patients, directed at alterations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 2 (FGFR2), and KIT proto-oncogene, receptor tyrosine kinase (KIT). Intrapatient, inter-CTC genomic heterogeneity was observed, at times between time points, in subclonal alterations. Our analysis suggests that serial monitoring of the CTC genome is feasible and should enable real-time tracking of tumor evolution during progression, permitting more combination precision medicine interventions

Bulletin officiel des contributions directes et du cadastre / Direction générale des impôts

19361936 (A1936,N7,PART2)

Comparative Molecular Analysis of Primary Central Nervous System Lymphomas and Matched Vitreoretinal Lymphomas by Vitreous Liquid Biopsy

Primary Central Nervous System Lymphoma (PCNSL) is a lymphoid malignancy of the brain that occurs in ~1500 patients per year in the US. PCNSL can spread to the vitreous and retina, where it is known as vitreoretinal lymphoma (VRL). While confirmatory testing for diagnosis is dependent on invasive brain tissue or cerebrospinal fluid sampling, the ability to access the vitreous as a proximal biofluid for liquid biopsy to diagnose PCNSL is an attractive prospect given ease of access and minimization of risks and complications from other biopsy strategies. However, the extent to which VRL, previously considered genetically identical to PCNSL, resembles PCNSL in the same individual with respect to genetic alterations, diagnostic strategies, and precision-medicine based approaches has hitherto not been explored. Furthermore, the degree of intra-patient tumor genomic heterogeneity between the brain and vitreous sites has not been studied. In this work, we report on targeted DNA next-generation sequencing (NGS) of matched brain and vitreous samples in two patients who each harbored VRL and PCSNL. Our strategy showed enhanced sensitivity for molecular diagnosis confirmation over current clinically used vitreous liquid biopsy methods. We observed a clonal relationship between the eye and brain samples in both patients, which carried clonal CDKN2A deep deletions, a highly recurrent alteration in VRL patients, as well as MYD88 p.L265P activating mutation in one patient. Several subclonal alterations, however, in the genes SETD2, BRCA2, TERT, and broad chromosomal regions showed heterogeneity between the brain and the eyes, between the two eyes, and among different regions of the PCNSL brain lesion. Taken together, our data show that NGS of vitreous liquid biopsies in PCNSL patients with VRL highlights shared and distinct genetic alterations that suggest a common origin for these lymphomas, but with additional site-specific alterations. Liquid biopsy of VRL accurately replicates the findings for PCNSL truncal (tumor-initiating) genomic alterations; it can also nominate precision medicine interventions and shows intra-patient heterogeneity in subclonal alterations. To the best of our knowledge, this study represents the first interrogation of genetic underpinnings of PCNSL with matched VRL samples. Our findings support continued investigation into the utility of vitreous liquid biopsy in precision diagnosis and treatment of PCNSL/VRL

Molecular Characterization of a Rare Case of Bilateral Vitreoretinal T Cell Lymphoma through Vitreous Liquid Biopsy

Vitreoretinal lymphoma (VRL) is an uncommon eye malignancy, and VRLs of T cell origin are rare. They are difficult to treat, and their molecular underpinnings, including actionable genomic alterations, remain to be elucidated. At present, vitreous fluid liquid biopsies represent a valuable VRL sample for molecular analysis to study VRLs. In this study, we report the molecular diagnostic workup of a rare case of bilateral T cell VRL and characterize its genomic landscape, including identification of potentially targetable alterations. Using next-generation sequencing of vitreous-derived DNA with a pan-cancer 126-gene panel, we found a copy number gain of BRAF and copy number loss of tumor suppressor DNMT3A. To the best of our knowledge, this represents the first exploration of the T cell VRL cancer genome and supports vitreous liquid biopsy as a suitable approach for precision oncology treatments