Kohli, Manish

PURPOSETumor tissue from metastatic castration-resistant prostate cancer (mCRPC) harbors frequent copy number variations (CNVs) in the PTEN-PI3K-AKT pathway. However, identifying CNVs in plasma cell–free DNA (cfDNA) has proven to be challenging. With emerging data supporting Akt inhibition in PTEN-deficient mCRPC, we profiled PTEN-PI3K-AKT pathway aberrations in patients with mCRPC using a novel cfDNA assay optimized for CNV detection.METHODSA next-generation sequencing–based cfDNA assay was used to profile 231 patients with mCRPC from two independent cohorts (Australian, n = 78; United States, n = 153). PTEN-PI3K-AKT pathway genomic aberrations were correlated with clinical outcomes, including progression-free survival and overall survival (OS).RESULTSPTEN loss and PIK3CA gain were detected in 37% (85 of 231) and 17% (39 of 231) of patients, respectively. Poorer outcomes were observed in patients with PTEN-PI3K-AKT pathway aberrations, including those with dual PTEN loss and PIK3CA gain (hazard ratio 2.3, 95% CI 1.2 to 4.4). Cumulative CNV burden in the PTEN-PI3K-AKT and androgen receptor (AR) pathways was associated with significantly worse clinical outcomes (0 v 1 v ≥ 2 CNVs in Australian cohort: median OS 33.5 v 17.2 v 9.7 months, P < .001; 0 v 1 v ≥ 2 CNVs in US cohort: median OS 35.5 v 14.3 v 9.2 months, P < .001). Notably, 21% (31 of 146) of PTEN-neutral patients harbored alternative PTEN-PI3K-AKT pathway aberrations.CONCLUSIONPTEN-PI3K-AKT pathway CNVs were readily detected using our cfDNA assay, with the prevalence of PTEN loss comparable with tissue-based studies. Additional PTEN-PI3K-AKT pathway aberrations were found in one fifth of PTEN-neutral cases. Concurrent CNVs in the PTEN-PI3K-AKT and AR pathways portended poor survival, and identifying this high-risk patient subset for dual AR/Akt inhibition may optimize precision treatment with Akt inhibitors in mCRPC.

256 Background: PARP inhibitors, olaparib and rucaparib recently improved survival outcomes (in the Profound and Triton-2 trials) in pts with aPC harboring HRRm. Notably, ~35% of pts screened for the PROfound trial did not meet eligibility solely due to the lack of adequate quality/quantity of tumor tissue required for CGP (De Bono, NEJM, 2020). cfDNA analysis non-invasively assesses tumor-derived genomic alterations. We evaluated the prevalence of HRRm in a real-world aPC population, who had commercially available cfDNA assay results available. Methods: cfDNA based CGP (using a clinically-validated 73- to 74-gene panel i.e. Guardant360 or G360) from consecutive aPC pts between 11/2016–8/2020 were used for detection of HRRm. Frameshift and nonsense mutations were included as pathogenic. Variants of unknown significance were excluded. In this unmatched study, the prevalence of each HRRm was compared with those reported in literature using the chi square test. Results: cfDNA CGP from 7701 aPC pts were available for interrogation of BRCA1/BRCA2 mutations, & from 4671 aPC pts for ATM mutations. Prevalence of BRCA1 and 2 as detected by cfDNA was similar to historical CGP of primary tissue. Prevalence of BRCA1 was higher than historical CGP of metastatic tissue. Prevalence of ATM in cfDNA was higher than historic tumor tissue CGP but similar to prior reports from cfDNA testing (Table). Conclusions: In this large real-world population of pts with aPC undergoing routine cfDNA CGP by a CLIA certified lab, prevalence of HRRm was similar (or higher for BRCA1 and ATM) to what has been previously reported from the tumor tissue. These data provide the rationale for utilizing cfDNA CGP as a routine test for detection of HRRm in men with aPC to identify men who are candidates for PARPi. [Table: see text]

159 Background: Tumour tissue from metastatic castration-resistant prostate cancer (mCRPC) harbors frequent copy number variations (CNVs) in the phosphatidylinositol-3-kinase (PI3K)/Akt-signaling pathway. However, identifying CNVs in plasma cell-free DNA (cfDNA) has proven challenging. With emerging data supporting Akt inhibition in PTEN-deficient mCRPC, cfDNA assays for robustly identifying PI3K/Akt pathway aberrations including CNVs are urgently required. Methods: In this multi-institutional prospective biomarker study, we used the Predicine cfDNA assay, optimised for CNV detection, to perform targeted sequencing in 231 mCRPC patients in two independent cohorts (Australian, n = 78; US, n = 153). Kaplan-Meier survival estimates and multivariable Cox regression analysis were used to assess associations between genomic aberrations and progression-free survival (PFS) and overall survival (OS). Results: PTEN loss and PIK3CA gain were detected in 37% (85/231) and 17% (39/231) of patients, respectively. Poorer outcomes were observed in patients with PI3K/Akt pathway aberrations, including those with dual PTEN loss and PIK3CA gain (HR 2.3, 95% CI 1.2-4.4). Similarly, cumulative CNV burden in the PI3K/Akt and AR pathways (0 vs 1 vs ≥2 CNVs in Australian cohort: median OS 33.5 vs 17.2 vs 9.7 months, p< 0.001; 0 vs 1 vs ≥2 CNVs in US cohort: median OS 35.5 vs 14.3 vs 9.2 months, p< 0.001) was associated with significantly worse clinical outcomes. Notably, 21% (31/146) of PTEN-neutral patients harbored other alterations in the PI3K/Akt pathway. Conclusions: Our cfDNA assay readily detected PI3K/Akt pathway CNVs, with the prevalence of PTEN loss comparable to prior tissue sequencing studies. CNVs in the PI3K/Akt pathway were associated with deleterious clinical outcomes, especially when concurrent with AR gain. Additional PI3K/Akt pathway aberrations were found in approximately one-fifth of PTEN-neutral mCRPC. Collectively, our data demonstrate the potential utility of profiling cfDNA to facilitate and optimize patient selection for treatment with Akt inhibitors in mCRPC. [Table: see text]

Background: Bone metastatic prostate cancer (BMPCa), despite the initial responsiveness to androgen deprivation therapy (ADT), inevitably becomes resistant. Recent clinical trials with upfront treatment of ADT combined with chemotherapy or novel hormonal therapies (NHTs) have extended overall patient survival. These results indicate that there is significant potential for the optimization of standard-of-care therapies to delay the emergence of progressive metastatic disease. Methods: Here, we used data extracted from human bone metastatic biopsies pre- and post-abiraterone acetate/prednisone to generate a mathematical model of bone metastatic prostate cancer that can unravel the treatment impact on disease progression. Intra-tumor heterogeneity in regard to ADT and chemotherapy resistance was derived from biopsy data at a cellular level, permitting the model to track the dynamics of resistant phenotypes in response to treatment from biological first-principles without relying on data fitting. These cellular data were mathematically correlated with a clinical proxy for tumor burden, utilizing prostate-specific antigen (PSA) production as an example. Results: Using this correlation, our model recapitulated the individual patient response to applied treatments in a separate and independent cohort of patients (n = 24), and was able to estimate the initial resistance to the ADT of each patient. Combined with an intervention-decision algorithm informed by patient-specific prediction of initial resistance, we propose to optimize the sequence of treatments for each patient with the goal of delaying the evolution of resistant disease and limit cancer cell growth, offering evidence for an improvement against retrospective data. Conclusions: Our results show how minimal but widely available patient information can be used to model and track the progression of BMPCa in real time, offering a clinically relevant insight into the patient-specific evolutionary dynamics of the disease and suggesting new therapeutic options for intervention. Trial registration: NCT # 01953640. Funding: Funded by an NCI U01 (NCI) U01CA202958-01 and a Moffitt Team Science Award. CCL and DB were partly funded by an NCI PSON U01 (U01CA244101). AA was partly funded by a Department of Defense Prostate Cancer Research Program (W81XWH-15-1-0184) fellowship. LC was partly funded by a postdoctoral fellowship (PF-13-175-01-CSM) from the American Cancer Society.

Precise management of kidney cancer requires the identification of prognostic factors. hPG80 (circulating progastrin) is a tumor promoting peptide present in the blood of patients with various cancers, including renal cell carcinoma (RCC). In this study, we evaluated the prognostic value of plasma hPG80 in 143 prospectively collected patients with metastatic RCC (mRCC). The prognostic impact of hPG80 levels on overall survival (OS) in mRCC patients after controlling for hPG80 levels in non-cancer age matched controls was determined and compared to the International Metastatic Database Consortium (IMDC) risk model (good, intermediate, poor). ROC curves were used to evaluate the diagnostic accuracy of hPG80 using the area under the curve (AUC). Our results showed that plasma hPG80 was detected in 94% of mRCC patients. hPG80 levels displayed high predictive accuracy with an AUC of 0.93 and 0.84 when compared to 18–25 year old controls and 50–80 year old controls, respectively. mRCC patients with high hPG80 levels (>4.5 pM) had significantly lower OS compared to patients with low hPG80 levels (<4.5 pM) (12 versus 31.2 months, respectively; p = 0.0031). Adding hPG80 levels (score of 1 for patients having hPG80 levels > 4.5 pM) to the six variables of the IMDC risk model showed a greater and significant difference in OS between the newly defined good-, intermediate- and poor-risk groups (p = 0.0003 compared to p = 0.0076). Finally, when patients with IMDC intermediate-risk group were further divided into two groups based on hPG80 levels within these subgroups, increased OS were observed in patients with low hPG80 levels (<4.5 pM). In conclusion, our data suggest that hPG80 could be used for prognosticating survival in mRCC alone or integrated to the IMDC score (by adding a variable to the IMDC score or by substratifying the IMDC risk groups), be a prognostic biomarker in mRCC patients.

Abstract Background: Obesity is a well established risk factor not only for increased prostate cancer incidence, but for poorer survival from prostate cancer. However, there is limited knowledge on the role of the circulating lipidome in prostate cancer. In order to target lipid metabolism optimally, it is critical to understand the spectrum of changes in circulating lipid profiles and the association with clinical outcome across the natural history of prostate cancer. Aim: To assess the relationship between the plasma lipidome and clinical outcome in localized and metastatic prostate cancer. Methods: Liquid chromatography-tandem mass spectrometry was used to quantitate over 500 lipid species in plasma samples from 3 cohorts (1) 389 men with localized prostate cancer, (2) 44 men with metastatic hormone-sensitive prostate cancer (mHSPC), and (3) 137 men with metastatic castration-resistant prostate cancer (CRPC). Associations between circulating lipids with metastatic relapse, androgen-deprivation therapy (ADT) failure or overall survival for each relevant disease stage were examined by latent class analysis and cox regression. Results: Circulating lipid profiles displaying elevated levels of ceramides were associated with metastatic relapse in localized prostate cancer (HR 5.8, 95% CI 3.0-11, P 1 × 10−6), early ADT failure in mHSPC (HR 2.4, 95% CI 1.1-5.3, P 0.03), and shorter overall survival in CRPC (HR 2.5, 95% CI 1.7-3.7, P 2 × 10−6). ADT failure in mHSPC and shorter overall survival in CRPC were also associated with elevated levels of other sphingolipids (sphingomyelins, hexosylceramides). The prognostic significance of the high risk lipid profiles in localized prostate cancer was independent of clinicopathological characteristics (lipid profile HR 4.7, 95%CI 2.4-9.3, P 7 × 10−6) when modeled with Gleason score (P&lt;0.0001) and pathological stage (P&lt;0.0001). Furthermore, the circulating lipid profiles were independent of metabolic factors (localized prostate cancer lipid profile HR 8.2, 95% CI 2.6-25, P 0.0003 when modeled with diabetes, statin, hypertension, body mass index [BMI]; CRPC lipid profile HR 2.6, 95% CI 1.7-3.8, P x10−6 when modeled with BMI). Conclusion: Elevated circulating ceramides are associated with poorer clinical outcomes across the natural history of prostate cancer (from localized to metastatic hormone-sensitive to metastatic castration resistant prostate cancer). This demonstrates that aberrant lipid metabolism in the patients occurs early in prostate cancer and could be therapeutically targeted in prospective clinical trials to improve prostate cancer outcomes. Citation Format: Lisa Glen Horvath, Hui-Ming Lin, Blossom Mak, Kate Mahon, Nicole Yeung, Maria Docanto, Peter Sutherland, andrew Shepherd, Winston Tan, Arun Azad, Manish Kohli, Peter Meikle, Lisa Butler. Lipidomic analysis of circulating lipids across the natural history of prostate cancer identifies aberrant ceramide metabolism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4761.

Abstract Background: Androgen receptor (AR) amplification is a common genomic event in advanced prostate cancer, in particular, the metastatic castration-resistant prostate cancer (mCRPC) and is associated with poor treatment response and short overall survival (OS). Recent studies also show more frequent amplification at AR enhancer region in the mCRPC patients. The aim of this study is to evaluate clinical utility of multiplex digital PCR assay to detect amplification at AR locus in cell-free DNA (cfDNA) for prognostication of mCRPC. Methods: Plasma samples were collected from 108 patients with mCRPC before stage-specific treatment. The target regions were selected based on our previous sequencing data showing frequent amplification in mCRPC patients at AR loci, including AR enhancer, AR (AR exon 1 and exon 8) and OPHN1 (downstream of AR). Multiplex digital PCR assays to detect four targets were developed by adjusting the concentrations of primers and probes. The Kaplan-Meier (KM) estimate was used for OS analysis. Results: We first examined existing databases to determine relative stable genomic regions in prostate cancer. This analysis showed most stable regions in three gene loci including C2orf16 at 2p23.2, FAM111B at 11q12.1, and GRIA3 at Xq25, which were then used as reference controls for copy number normalization. We then designed two multiplex digital PCR assays to quantify copy number of selected targets. To separate two different targets with same fluorescence, we adjusted ratio of FAM-labeled probes with 1: 0.4 and HEX-labeled probes with 1:0.4. The ratio of FAM to HEX probes was 1:1.5 in final assays. The combination of four different targets in one reaction gave 9 possible clusters in the 2D amplitude space. We defined the amplification as the normalized ratio ≥ 1.8 between target and reference loci. By applying this cutoff in 108 mCRPC samples, we detected the amplification of AR-enhancer, AR and OPHN1 in 38, 34, and 25 samples, respectively. KM analysis showed the significant association of amplification in AR-enhancer (p=0.0053, HR=2.16[1.41-6.53]), AR (p=0.04, HR=1.74[1.04-4.03]) and OPHN1 (p=0.031, HR=1.91[1.07-5.53]) with OS. This result was consistent with whole genome sequencing data in the same group of patients. Conclusions: We established a highly sensitive multiplex digital PCR assay to detect copy number changes of AR, AR enhancer and OPHN1 in plasma cfDNA and demonstrated its potential clinical use as non-invasive testing tool for mCRPC prognosis. The easy-to-use and low-cost multiplex digital PCR assay covers multiple gene loci, which can aid in improving the sensitivity of detecting amplification at AR locus in the highly heterogeneous mCRPC patients. Citation Format: Meijun Du, Chiang-Ching Huang, Winston Tan, Manish Kohli, Liang Wang. Clinical utility of multiplex digital PCR to detect androgen receptor amplification in cell-free DNA for prognosis of metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4319.

Blood is a readily accessible biofluid containing a plethora of important proteins, nucleic acids, and metabolites that can be used as clinical diagnostic tools in diseases, including cancer. Like the on-going efforts for cancer biomarker discovery using the liquid biopsy detection of circulating cell-free and cell-based tumor nucleic acids, the circulatory proteome has been underexplored for clinical cancer biomarker applications. A comprehensive proteome analysis of human serum/plasma with high-quality data and compelling interpretation can potentially provide opportunities for understanding disease mechanisms, although several challenges will have to be met. Serum/plasma proteome biomarkers are present in very low abundance, and there is high complexity involved due to the heterogeneity of cancers, for which there is a compelling need to develop sensitive and specific proteomic technologies and analytical platforms. To date, liquid chromatography mass spectrometry (LC-MS)-based quantitative proteomics has been a dominant analytical workflow to discover new potential cancer biomarkers in serum/plasma. This review will summarize the opportunities of serum proteomics for clinical applications; the challenges in the discovery of novel biomarkers in serum/plasma; and current proteomic strategies in cancer research for the application of serum/plasma proteomics for clinical prognostic, predictive, and diagnostic applications, as well as for monitoring minimal residual disease after treatments. We will highlight some of the recent advances in MS-based proteomics technologies with appropriate sample collection, processing uniformity, study design, and data analysis, focusing on how these integrated workflows can identify novel potential cancer biomarkers for clinical applications.

Amplification of androgen receptor (AR) is a common genomic event in metastatic castration-resistant prostate cancer (mCRPC). To evaluate the prognostic value of the amplifications of specific loci in the AR gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted AR enhancer (AR-En), AR exon 1 (AR-E1), AR exon 8 (AR-E8) and OPHN1 (downstream of AR). We selected three relatively stable genes, C2orf16, FAM111B, and GRIA3, as reference controls for copy number normalization. One hundred and eight mCRPC patients were recruited to test the association of specific AR loci amplification with clinical outcome. Using a normalized ratio ≥ 1.92 as cutoff, amplification of AR-En, AR-E1, AR-E8 and OPHN1 was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with AR region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at AR-En, AR-E1, and AR-E8. Six (14.6%) patients showed independent AR-En amplification, while the remaining 3 (7.3%) demonstrated AR-E8 amplification only. Kaplan–Meier analysis showed overall survival’s association with the amplification of AR-En (p = 0.02, HR = 1.68 (1.07–2.65)), AR-E8 (p = 0.02, HR = 1.78 (1.08–2.92)) and AR-En-E8 (the combination of AR-En and AR-E8 (p = 0.009, HR = 1.77 (1.15–2.73)). Multivariate models that included AR-En-E8 amplification and clinical factors significantly improved prognostic performance (p = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.

5583 Background: FDA has approved three novel AAs [Apalutamide(A), Darolutamide(D) and Enzalutamide(E)] in combination with Androgen deprivation therapy ( ADT) for treatment of (nmCRPC) patients (pts). We report the cost-effectiveness of these drugs from the US perspective to help facilitate the choice of these agents for clinical practice. Methods: A life time Markov state-transition model was constructed with three health states (Metastasis-Free Survival[MFS], Metastatic disease, and Death) to compare cost-effectiveness of AA therapies for treatment of nmCRPC based on US healthcare payer perspective. A network meta-analysis of MFS and OS was conducted due to the lack of head to head trials. An approximation of the original individual-level patient time-to-event data were derived from digitized Kaplan-Meier curves for OS and MFS. Weibull distributions was selected as the best fitted model fitted and extrapolated as per the NICE decision support unit recommendations. Medication costs were based on wholesale acquisition cost. Adverse event (AE) grades 3/4 management costs were incorporated in the model. Discount rate of 3% per year was applied to costs and effects. Life years (LYs) and quality adjusted life years (QALYs) for each treatment as well as the incremental cost effectiveness (ICER) and cost utility (ICUR) ratios were estimated. Base case analyses (BCA) and probabilistic sensitivity analyses (PSA) were estimated. Results: The table summarizes the results form BCA analyses. A+ADT offers best gain in LYs (8.37yrs) and QALYs (5.30 yrs) but at higher cost. Conclusions: Apalutamide was associated with gains in LYs and QALYs traded off with higher lifetime cost relative to other AA alternatives. ADT was associated with lower gains in LYs and QALYs traded off with lower lifetime cost relative to other alternatives. Based on a $150,000/QALY threshold pay off, A+ADT is likely more cost effective compared to E+ADT or ADT alone; while E+ ADT may be more cost effective compared to D+ ADT. [Table: see text]