Localized prostate cancers are genetically variable and frequently multifocal, comprising spatially distinct regions with multiple independently-evolving clones. To date there is no understanding of ...whether this variability can influence management decisions for patients with prostate tumors. Here, we present a single case from a clinical trial of neoadjuvant intense androgen deprivation therapy. A patient was diagnosed with a large semi-contiguous tumor by imaging, histologically composed of a large Gleason score 9 tumor with an adjacent Gleason score 7 nodule. DNA sequencing demonstrates these are two independent tumors, as only the Gleason 9 tumor harbors single-copy losses of PTEN and TP53. The PTEN/TP53-deficient tumor demonstrates treatment resistance, selecting for subclones with mutations to the remaining copies of PTEN and TP53, while the Gleason 7 PTEN-intact tumor is almost entirely ablated. These findings indicate that spatiogenetic variability is a major confounder for personalized treatment of patients with prostate cancer.
Patients diagnosed with high risk localized prostate cancer have variable outcomes following surgery. Trials of intense neoadjuvant androgen deprivation therapy (NADT) have shown lower rates of ...recurrence among patients with minimal residual disease after treatment. The molecular features that distinguish exceptional responders from poor responders are not known.
To identify genomic and histologic features associated with treatment resistance at baseline.
Targeted biopsies were obtained from 37 men with intermediate- to high-risk prostate cancer before receiving 6 mo of ADT plus enzalutamide. Biopsy tissues were used for whole-exome sequencing and immunohistochemistry (IHC).
We assessed the relationship of molecular features with final pathologic response using a cutpoint of 0.05 cm3 for residual cancer burden to compare exceptional responders to incomplete and nonresponders. We assessed intratumoral heterogeneity at the tissue and genomic level, and compared the volume of residual disease to the Shannon diversity index for each tumor. We generated multivariate models of resistance based on three molecular features and one histologic feature, with and without multiparametric magnetic resonance imaging estimates of baseline tumor volume.
Loss of chromosome 10q (containing PTEN) and alterations to TP53 were predictive of poor response, as were the expression of nuclear ERG on IHC and the presence of intraductal carcinoma of the prostate. Patients with incompletely and nonresponding tumors harbored greater tumor diversity as estimated via phylogenetic tree reconstruction from DNA sequencing and analysis of IHC staining. Our four-factor binary model (area under the receiver operating characteristic curve AUC 0.89) to predict poor response correlated with greater diversity in our cohort and a validation cohort of 57 Gleason score 8–10 prostate cancers from The Cancer Genome Atlas. When baseline tumor volume was added to the model, it distinguished poor response to NADT with an AUC of 0.98. Prospective use of this model requires further retrospective validation with biopsies from additional trials.
A subset of prostate cancers exhibit greater histologic and genomic diversity at the time of diagnosis, and these localized tumors have greater fitness to resist therapy.
Some prostate cancer tumors do not respond well to a hormonal treatment called androgen deprivation therapy (ADT). We used tumor volume and four other parameters to develop a model to identify tumors that will not respond well to ADT. Treatments other than ADT should be considered for these patients.
A subset of patients present with high-risk localized prostate cancer that exhibits greater histologic and genomic diversity. These patients are less likely to respond to intense neoadjuvant androgen deprivation therapy.
Neoadjuvant intense androgen deprivation therapy (iADT) can exert a wide range of histological responses, which in turn are reflected in the final prostatectomy specimen. Accurate identification and ...measurement of residual tumor volumes are critical for tracking and stratifying patient outcomes.
The goal of this current study was to evaluate the ability of antibodies against prostate-specific membrane antigen (PSMA) to specifically detect residual tumor in a cohort of 35 patients treated with iADT plus enzalutamide for 6 months prior to radical prostatectomy.
Residual carcinoma was detected in 31 patients, and PSMA reacted positively with tumor in all cases. PSMA staining was 96% sensitive for tumor, with approximately 82% of benign regions showing no reactivity. By contrast, PSMA positively reacted with 72% of benign regions in a control cohort of 37 untreated cases, resulting in 28% specificity for tumor. PSMA further identified highly dedifferentiated prostate carcinomas including tumors with evidence of neuroendocrine differentiation.
We propose that anti-PSMA immunostaining be a standardized marker for identifying residual cancer in the setting of iADT.
Localized prostate cancer develops very slowly in most men, with the androgen receptor (AR) and MYC transcription factors amongst the most well-characterized drivers of prostate tumorigenesis. ...Canonically, MYC up-regulation in luminal prostate cancer cells functions to oppose the terminally differentiating effects of AR. However, the effects of MYC up-regulation are pleiotropic and inconsistent with a poorly proliferative phenotype. Here we show that increased MYC expression and activity are associated with the down-regulation of MEIS1, a HOX-family transcription factor. Using RNA-seq to profile a series of human prostate cancer specimens laser capture microdissected on the basis of MYC immunohistochemistry, MYC activity, and MEIS1 expression were inversely correlated. Knockdown of MYC expression in prostate cancer cells increased the expression of MEIS1 and increased the occupancy of MYC at the MEIS1 locus. Finally, we show in laser capture microdissected human prostate cancer samples and the prostate TCGA cohort that MEIS1 expression is inversely proportional to AR activity as well as HOXB13, a known interacting protein of both AR and MEIS1. Collectively, our data demonstrate that elevated MYC in a subset of primary prostate cancers functions in a negative role in regulating MEIS1 expression, and that this down-regulation may contribute to MYC-driven development and progression.
Abstract
Background and Hypothesis
Cancer cells release circulating tumor DNA (ctDNA) into the bloodstream, which has the potential to be clinically relevant as a marker of tumor clonality and a ...marker of clinical response. This "liquid biopsy" may provide a non-invasive approach to cancer care diagnosis and monitoring. We have optimized a PCR assay for rapid and inexpensive selection and detection of a priori sequenced targets, serving as clonal and subclonal markers of cancer evolution. Our hypothesis is that this assay will allow us to detect circulating free DNA and ctDNA while modeling tumor clonality and measuring clinical response in patients undergoing treatment for metastatic prostate cancer.
Methods
From a given panel of somatic point mutations previously identified via unbiased sequencing of matched tumor and normal samples, multiplex PCR primer mixes were generated using the IonTorrent AmpliSeq Design tool, creating locus-specific primers flanking each point mutation by approximately 60bp on each side. To the 5’ end of each forward primer, a 7-base degenerate unique molecular identifier (UMI) was synthesized, to which an additional 32-base sequence complementary to the forward Illumina Nextera adaptor was further added. To the 3’ end of the reverse primer, a 34-base sequence complementary to the reverse Illumina Nextera adaptor was added. Forward primers for each patient-specific set were pooled and hybridized to aliquots of ctDNA extracted from patient plasma. The tagged product was amplified in exponential PCR with the reverse primer pool and a full-length Nextera i7 Indexing primer. Following purification and size selection, the tagged and partial-adaptor-ligated library was amplified in additional exponential cycles of PCR with full length Nextera i5 and i7 indexing primers. Libraries were then quantified and sequenced via MiSeq.
Results and Conclusions
We have previously shown that for a series of seven patients, primers designed against clonal and subclonal mutations successfully amplified their genomic targets in 269 out of 280 amplicons, averaging 10 amplicons per library and 100,000x target coverage per amplicon. Following duplicate reduction, approximately 1,000 unique molecules were sequenced. Spike-in experiments were utilized to establish the lower limit of reliable detection. This assay is currently being applied to patients with metastatic prostate cancer who had blood drawn across multiple timepoints. We are currently analyzing these samples to model tumor clonality and treatment response across multiple patients.
Relevance and Importance
We have developed a robust, patient-specific assay for detection of ctDNA and monitoring patients over time. This assay has the potential to provide a patient-specific and non-invasive approach for monitoring treatment and remission of metastatic prostate cancer.
Citation Format: Nicholas T. Terrigino, S. Thomas Hennigan, Shana Trostel, Scott C. Wilkinson, Huihui Ye, Adam G. Sowalsky. Development of an allele-specific assay for detecting circulating tumor DNA in prostate cancer 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 736.
Abstract
Background: The zinc-finger homeobox ZFXH3 (also known as ATBF1 for AT motif-binding factor 1) is a transcription factor that is frequently downregulated or deleted in human prostate ...cancers. In a recent clinical trial (NCT02430480), patients with localized prostate cancer (PCa) received six months of neoadjuvant androgen deprivation therapy (ADT) with 10.8mg of the GnRH agonist goserelin every 12 weeks and 160mg of oral enzalutamide daily before a radical prostatectomy (RP). Throughout the six months, the patients had two multiparametric MRIs (mpMRI) at baseline and prior to surgery, along with targeted biopsies of the tumor extracted. DNA from laser capture microdissection (LCM) biopsy and RP tissues was extracted for whole exome sequencing with somatic mutation calling and copy number analysis. The most common alteration in all residual tumor samples shared a single-copy loss or deletion of chromosome 16q overlapping with ZFHX3. We hypothesized that ZFHX3 is necessary for the proper development of normal human prostates and drives tumor-suppressive activity. Methods: To mimic the reduced expression of ZFHX3 observed in patient samples, we first surveyed ZFHX3 expression by Western blotting and real-time qPCR and then developed shRNA knockdowns in androgen-sensitive (22Rv1 and LNCaP) and insensitive (C4-2) PCa cell lines. These cell lines were challenged by dose-response curve studies utilizing standard fetal bovine serum (FBS) growth media with the androgen receptor (AR) axis inhibitors abiraterone and enzalutamide. Continued treatment conditions included charcoal dextran-stripped fetal calf serum (CSS) to simulate androgen deprivation supplemented with either abiraterone or enzalutamide. Cell proliferation was determined at the end of the five-day dose-response curve treatment studies using the CellTiter Glo 2.0 cell viability assay. Results and Conclusions: In the three PCa cell lines with ZFHX3 knockdown, we observed increased sensitivity to AR-targeting drugs with a lower IC50 dose. In addition, ZFHX3 knockdown cell lines exhibited increased cell proliferation compared to scrambled control and parental cell lines using dose-response time course studies. These preliminary results form the scientific premise for this work and substantiate ongoing efforts to expand these assays to more cell lines and treatments. Our future efforts will provide mechanistic insights into the functions of ZFHX3 through chromatin immunoprecipitation sequencing with antibodies against AR and FOXA1 and the expansion of ZFHX3 knockdown into additional cell lines to investigate the transcriptional activity of multiple hormonal signaling pathways. We will further explore genomic interactions between the effects of ZFHX3 reduction and alterations to commonly-mutated genes in PCa, such as PTEN, TP53, and SPOP. Findings in this study will seek to determine how ZFHX3 exerts its tumor suppressor function and its role in the development and progression of prostate cancer.
Citation Format: Isaiah M. King, John R. Bright, Nicholas T. Terrigino, Scott Wilkinson, Adam G. Sowalsky. Investigating the function of ZFHX3 in hormone sensitive prostate cancer initiation and treatment resistance abstract. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A004.
Abstract
Background: Molecular characterization of mCRPC has revealed disruption of DNA damage repair genes that may improve sensitivity to PARP inhibitors such as olaparib (O), which in turn may ...further complement the activity of the PD-L1 immune checkpoint inhibitor, durvalumab (D), through mechanisms such as activation of the STING pathway. We hypothesized that (epi)genomic analyses of circulating tumor DNA (ctDNA) would identify mechanisms of treatment response in patients with mCRPC treated with O plus D (NCT02484404). Methods: Eligible patients for this study had mCRPC with prior treatment by abiraterone and/or enzalutamide. Study participants were administered 300 mg of O twice daily and 1500 mg of D every 4 weeks. We obtained blood samples in Streck tubes from 38 (out of 60) study participants. Plasma samples were acquired at baseline and at progression. Buffy coat was isolated from each sample. Genomic DNA and cell-free DNA (cfDNA) were separately isolated and assembled into sequencing libraries. Low-pass whole-genome sequencing (3-4 × coverage) was performed for somatic copy number and fragmentomic analyses. In a subset of samples with the greatest cfDNA content, chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against H3K4me2 and H3K4me3 was performed directly in additional aliquots of plasma. Results: Amongst the 38 patients with at least one cfDNA sample, 5 patients had a radiographic partial response (PR) per RECIST v1.1, 28 patients had stable disease (SD), and 6 patients had progressive disease (PD) during the first two months of treatment. At baseline, ctDNA content in plasma was significantly lower in patients who went on to have PR or SD versus PD (p=0.02). However, upon progression, ctDNA content in plasma was not significantly different between groups. Losses to PTEN and NKX3-1 were significantly enriched in ctDNA from patients with PD (p=0.041 and p=0.047, respectively). By contrast, biallelic combined losses to BRCA2, RB1, BRCA1 and TP53 were observed in all PRs at baseline but not in any patient with PD (p=0.048). In patients with SD, longitudinal fragmentomic analysis inferred that the activity of the mitogenic KLF4 transcription factor (TF) was the most increased upon progression, while activity of the protective HIF2A TF was the most decreased. However, the TF binding motifs predicted by H3K4me2 and H3K4me3 cfDNA ChIP-seq did not significantly change in patients with PD. Conclusions: Distinctive genomic and epigenomic profiles were observed in plasma from patients with responsive vs. progressive disease, and the profiles may predict responses to combination D+O therapy. Deeper whole-genome sequencing of these samples currently in progress will reveal the contribution of germline and somatic point mutations to clinical response. Ongoing efforts to estimate the phylogenetic architecture of patients’ tumors from these plasma samples will enable the inference of drug response phenotypes as a function of evolutionary potential and potentially identify new opportunities to target treatment-resistant disease.
Citation Format: Anna Baj, Clara C. Y. Seo, Nicholas T. Terrigino, John R. Bright, S. Thomas Hennigan, Isaiah M. King, Shana Y. Trostel, William D. Figg, William L. Dahut, Jung-Min Lee, David Y. Takeda, Fatima Karzai, Adam G. Sowalsky. Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition abstract. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A053.
Abstract
Background: Intense neoadjuvant androgen deprivation therapy (inADT) reduces prostate cancer recurrence in approximately 40% of patients with intermediate/high risk prostate cancer. Using ...samples from our recent phase 2 study (NCT02430480) from patients undergoing 6 months of inADT prior to radical prostatectomy, we sought to examine molecular features present at baseline that correlate with pathologic response following surgery. Methods: Tumors from 37 patients with locally advanced prostate cancer were subjected to immunohistochemistry (IHC) and laser capture microdissection to obtain 143 histologically distinct foci. RNA was isolated from each focus and used to generate RNA-seq libraries. Differentially-expressed genes were determined per-unit of residual tumor volume using linear mixed-effect models. Histologic features and staining intensities were obtained from IHC of posttreatment specimens. Ingenuity Pathway Analysis (IPA) identified upstream regulators at baseline that interacted with pathologic outcome. Cancer cell lines were treated with a panel of EGFR family inhibitors and androgen receptor (AR) axis inhibitors. Protein and RNA expression were measured to identify the impact of dual inhibition. Results: In pre-treatment targeted biopsies from men with locally advanced prostate cancer, we observed that tumor samples that exhibited worse response to inADT had lower AR activity at baseline, presumably due to a decreased dependency on AR, potentially related to increased dependency on alternative growth pathways. These incomplete/nonresponding tumors displayed a significant increase in expression of EFGR family signaling (EGFR, HER2, and HER3) using IPA, which have previously been shown to serve as bypass mechanisms to AR signaling to drive downstream PI3K activity. HER2 was the most abundantly expressed protein by IHC in posttreatment tumors. Given the multitude of receptor tyrosine kinases (RTKs) regulated by EGFR family signaling and RTK inhibitors targeting this activity, we examined the impact of combining RTK inhibitors with ADT to overcome resistance to ADT. Afatinib, lapatinib, and neratinib showed the greatest anti-tumor efficacy in vitro. Treatment with these agents rapidly and dynamically increased AR expression and activity. We observed synergistic effects on cell viability and proliferation by treating cell lines with combination therapy of antiandrogen therapy and these inhibitors. Conclusions: Our data suggest that elevated HER2 activity defines an AR-low subtype of prostate cancer that is resistant to inADT. Disrupting the HER2-mediated bypass of AR blockade re-sensitized resistant PC cells to AR-targeted therapy. These effects were observed in an untreated setting and represent a divergent path in tumorigenesis where prostate tumors develop with intrinsically lower AR activity. These findings will be validated using genetic abrogation of HER2 in additional clinical samples, organoids, and patient-derived xenograft models, and represent new opportunities for combination therapy in newly-diagnosed high-risk disease.
Citation Format: Scott Wilkinson, Anson Ku, Isaiah King, Anna Baj, Daniel Low, Huihui Ye, Stephanie A. Harmon, Nicholas T. Terrigino, John R. Bright, Rayann Atway, Shana Y. Trostel, Rosina T. Lis, Baris Turkbey, William L. Dahut, Fatima Karzai, Adam G. Sowalsky. Examining the role of HER2 signaling in promoting prostate cancer proliferation in an androgen receptor-low environment abstract. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B021.
Abstract
Human cancer tissues are known to release their own DNA into body fluids such as plasma. These DNA molecules derived from tumors, collectively known as circulating tumor DNA (ctDNA), contain ...genetic information about disease progression at the time of sampling. Importantly, ctDNA has recently been utilized for capturing the whole-body disease profile and when sampled at various time points over the treatment, can inform the dynamic of clonal heterogeneity and its contribution to therapy failures. Metastatic castration-resistant prostate cancer (mCRPC) is a class of cancerous diseases with a high degree of clonal heterogeneity and high mortality which is largely due to the frequent emergence of therapy resistance. Therefore, reconstructing the clonal history for mCRPC diseases using ctDNA has great potential in identification of novel and targetable molecular mechanisms that drive mCRPC progression. To assess this possibility, we hypothesize that tumor clones that are responsible for therapy resistance carry distinct genomic profiles, which are captured in plasma ctDNA and could be computationally resolved through genomic sequencing and clonal reconstruction. To address this question, we obtained plasma cell-free DNA (cfDNA), a mixture of ctDNA and other tissue-derived DNA, from 38 individuals treated with combination PD-L1 and PARP inhibition involved in a recent clinical trial (NCT02484404). Whole-genome sequencing was performed using cfDNA and fragmented buffy coat DNA as germline control. cfDNA samples with a tumor fraction of lower than 10% were excluded. Various computational strategies were used to model the clonal structures of diseases and estimate the temporal order in which small and large structural variants emerged. Through this approach, we observed a negative association between cfDNA tumor fraction and therapy response. Multiple clonal evolutionary patterns leading to therapy failures were observed. For example, both gain and loss of activating androgen receptor mutations were detected in different cases following the treatment, which represents two distinct mechanisms of evading immunotherapy targeting. As a result, changes in the relevant transcriptional activities were also detected through ctDNA fragment-based computational inference. Additionally, clonal persistence was detected in multiple cases that exhibited stable diseases upon the treatment, which involves loss-of-function mutations in p53, ATM, and CDK12. To estimate the potential contribution to plasma ctDNA by metastatic diseases, multi-region whole genome and exome sequencing data of the tumors that are available for some cases prior to the treatment were also analyzed. Clones resolved from analyzing ctDNA were computationally mapped to the primary disease or metastatic tissue with a shared set of mutations in a subset of cases and may explain the patterns of treatment response. Our novel findings from this study will fill in a critical gap of knowledge about the complex genetic mechanisms driving mCRPC immunotherapy resistance.
Citation Format: Chennan Li, Anna Baj, Clara C. Y. Seo, Nicholas T. Terrigino, John R. Bright, S. Thomas Hennigan, Isaiah M. King, Scott Wilkinson, Shana Y. Trostel, William D. Figg, William L. Dahut, Jung Min Lee, David Y. Takeda, Fatima Karzai, Adam G. Sowalsky. Tracing the clonal dynamic of metastatic castration-resistant prostate cancer over immunotherapy using circulating tumor DNA abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR005.
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