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PURPOSE: Neoadjuvant intense androgen deprivation therapy (iADT) can exert a wide range of histologic 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. MATERIALS AND METHODS: 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 six months prior to radical prostatectomy. RESULTS: 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. CONCLUSIONS: We propose that anti-PSMA immunostaining be a standardized marker for identifying residual cancer in the setting of neoadjuvant intense androgen deprivation therapy.

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.

PURPOSE Despite decreased screening-based detection of clinically insignificant tumors, most diagnosed prostate cancers are still indolent, indicating a need for better strategies for detection of clinically significant disease before treatment. We hypothesized that patients with detectable circulating tumor DNA (ctDNA) were more likely to harbor aggressive disease. METHODS We applied ultra-low-pass whole-genome sequencing to profile cell-free DNA from 112 patients diagnosed with localized prostate cancer and performed targeted resequencing of plasma DNA for somatic mutations previously identified in matched solid tumor in nine cases. We also performed similar analyses of data from patients with metastatic prostate cancer. RESULTS In all cases of localized prostate cancer, even in clinically high-risk patients who subsequently had recurrent disease, ultra-low-pass whole-genome sequencing and targeted resequencing did not detect ctDNA in plasma acquired before surgery or before recurrence. In contrast, using both approaches, ctDNA was detected in patients with metastatic prostate cancer. CONCLUSION Our findings demonstrate clear differences between localized and advanced prostate cancer with respect to the dissemination and detectability of ctDNA. Because allele-specific alterations in ctDNA are below the threshold for detection in localized prostate cancer, other approaches to identify cell-free nucleic acids of tumor origin may demonstrate better specificity for aggressive disease.

Neoadjuvant intense androgen deprivation therapy can exert a wide range of histologic 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 detect residual tumor in a cohort of 35 patients treated with androgen deprivation therapy plus enzalutamide for six months prior to radical prostatectomy. Residual carcinoma was detected in 31 patients, and PSMA reacted positively with tumor in all cases. PSMA staining was 95.5% sensitive for tumor, with approximately 81.6% of benign regions showing no reactivity. By contrast, PSMA positively reacted with 72.2% of benign regions in a control cohort of 37 untreated cases, resulting in 27.8% 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 neoadjuvant intense androgen deprivation therapy.

Background 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. Objective To identify genomic and histologic features associated with treatment resistance at baseline. Design, setting, and participants 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). Outcome measurements and statistical analysis 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. Results and limitations 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. Conclusions 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. Patient summary 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.

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., Prostate cancer is often a multifocal disease but how best to manage this clinically remains unclear. Here, the authors report a single case study of a patient with two genetically diverse tumours which showed differential response to therapy.

368 Background: PCa is driven by androgen receptor (AR) signaling and neoadjuvant therapy with AR inhibitors offer an opportunity to improve cure rates in high-risk PCa particularly with utilization of multiparametric MRI (mpMRI). A loss of AR-regulated lineage characteristics and genomic loss of tumor suppressors RB1 and TP53 or mutations in DNA damage repair (DDR) genes can represent aggressive prostate variants. We conducted a feasibility study using mpMRI to evaluate tumor responses and resistance in newly diagnosed, high-risk PCa (NCT02430480). Methods: Pts were treated with androgen deprivation therapy (ADT) + enzalutamide (enza) 160 mg daily for 6 months (mos). Pts underwent 2 mpMRIs: baseline and post 6 mos treatment (trt). Post-trt mpMRI was followed by radical prostatectomy (RP). Primary endpoint: feasibility of mpMRI for localization and detection of PCa before and after ADT + enza. Results: 39 pts were enrolled on-study with 36 pts completing 6 mos trt and undergoing RP. Of 39 pts, 3 had disease progression. Conclusions: Neoadjuvant intense testosterone lowering therapy shows activity in PCa but a subset of pts not respond to AR-targeted therapies through lineage plasticity enabled by characteristic loss of RB1 and TP53 or due to genetic alterations. Identification of this high-risk patient population, along with development of treatment options, needs further investigation. Clinical trial information: NCT02430480. [Table: see text]

342 Background: The differential aggressiveness of potentially independent prostate cancer clones remains largely unknown. Appropriate prostate cancer staging using mpMRI and biopsy tissue can be confounded by sampling error. To date, there has been no understanding of whether clonal variability influences management decisions for localized prostate tumors. We sought to identify the sensitivity and genomic profile of distinct localized tumors from a patient following systemic intense neoadjuvant androgen deprivation therapy (ADT). Methods: A 66-year-old man with high risk prostate cancer enrolled in a Phase 2 study of intense neoadjuvant ADT (goserelin + enzalutamide; inADT). Baseline mpMRI showed a single semi-contiguous lesion encompassing the right apical-mid PZ extending into the left distal apical PZ. MR/US-fusion targeted biopsy was performed before 6 months of inADT. A second mpMRI was performed before radical prostatectomy. Whole exome sequencing on microdissected tumor foci identified somatic mutations and copy number alterations, which were further used with immunohistochemistry to assess tumor clonal architecture and genomic/phenotypic evolution of treatment resistant tumor. Results: We found two clonally independent tumors exhibited intrinsic heterogeneity at baseline which correlated with response or resistance. Biopsies of distinct left- and right-sided tumors showed differing histologies. mpMRI and pathology showed near complete response of the left-sided tumor and substantial resistance of the right-sided tumor, which exhibited a large intraductal component. Histology and whole exome data highlighted a divergence in the status of PTEN and TP53, tumor suppressor genes implicated in prostate cancer progression. Conclusions: These data highlight that even nascent prostate cancer is heterogenous and neoadjuvant therapeutic strategies will need to consider this for clinical optimization. Evolutionary trajectories that resulted in tumor heterogeneity in this case likely contributed to our observation that two independent prostate tumor nodules with distinct genetic alterations responded differently to neoadjuvant intense ADT. Clinical trial information: NCT02430480.

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.