Inactivation of CDK12 Delineates a Distinct Immunogenic Class of Advanced Prostate Cancer.

Using integrative genomic analysis of 360 metastatic castration-resistant prostate cancer (mCRPC) samples, we identified a novel subtype of prostate cancer typified by biallelic loss of CDK12 that is mutually exclusive with tumors driven by DNA repair deficiency, ETS fusions, and SPOP mutations. CDK12 loss is enriched in mCRPC relative to clinically localized disease and characterized by focal tandem duplications (FTDs) that lead to increased gene fusions and marked differential gene expression. FTDs associated with CDK12 loss result in highly recurrent gains at loci of genes involved in the cell cycle and DNA replication. CDK12 mutant cases are baseline diploid and do not exhibit DNA mutational signatures linked to defects in homologous recombination. CDK12 mutant cases are associated with elevated neoantigen burden ensuing from fusion-induced chimeric open reading frames and increased tumor T cell infiltration/clonal expansion. CDK12 inactivation thereby defines a distinct class of mCRPC that may benefit from immune checkpoint immunotherapy.

Genomic correlates of clinical outcome in advanced prostate cancer.

Heterogeneity in the genomic landscape of metastatic prostate cancer has become apparent through several comprehensive profiling efforts, but little is known about the impact of this heterogeneity on clinical outcome. Here, we report comprehensive genomic and transcriptomic analysis of 429 patients with metastatic castration-resistant prostate cancer (mCRPC) linked with longitudinal clinical outcomes, integrating findings from whole-exome, transcriptome, and histologic analysis. For 128 patients treated with a first-line next-generation androgen receptor signaling inhibitor (ARSI; abiraterone or enzalutamide), we examined the association of 18 recurrent DNA- and RNA-based genomic alterations, including androgen receptor (AR) variant expression, AR transcriptional output, and neuroendocrine expression signatures, with clinical outcomes. Of these, only RB1 alteration was significantly associated with poor survival, whereas alterations in RB1, AR, and TP53 were associated with shorter time on treatment with an ARSI. This large analysis integrating mCRPC genomics with histology and clinical outcomes identifies RB1 genomic alteration as a potent predictor of poor outcome, and is a community resource for further interrogation of clinical and molecular associations.

Efficacy of systemic therapies in men with metastatic castration resistant prostate cancer harboring germline ATM versus BRCA2 mutations.

BACKGROUND: Among men with metastatic prostate cancer, about 10% have germline alterations in DNA damage response genes. Most studies have examined BRCA2 alone or an aggregate of BRCA1/2 and ATM. Emerging data suggest that ATM mutations may have distinct biology and warrant individual evaluation. The objective of this study is to determine whether response to prostate cancer systemic therapies differs between men with germline mutations in ATM (gATM) and BRCA2 (gBRCA2). METHODS: This is an international multicenter retrospective matched cohort study of men with prostate cancer harboring gATM or gBRCA2. PSA50 response (≥50% decline in prostate-specific antigen) was compared using Fisher's exact test. RESULTS AND LIMITATIONS: The study included 45 gATM and 45 gBRCA2 patients, matched on stage and year of germline testing. Patients with gATM and gBRCA2 had similar age, Gleason grade, and PSA at diagnosis. We did not observe differences in PSA50 responses to abiraterone, enzalutamide, or docetaxel in metastatic castration resistant prostate cancer between the two groups; however, 0/7 with gATM and 12/14 with gBRCA2 achieved PSA50 response to PARPi (p < .001). Median (95% confidence interval) overall survival from diagnosis to death was 10.9 years (9.5-not reached) versus 9.9 years (7.1-not reached, p = .07) for the gATM and gBRCA2 cohorts, respectively. Limitations include the retrospective design and lack of mutation zygosity data. CONCLUSIONS: Conventional therapies can be effective in gATM carriers and should be considered before PARPi, which shows limited efficacy in this group. Men with gATM mutations warrant prioritization for novel treatment strategies.

Clinical determinants for successful circulating tumor DNA analysis in prostate cancer.

BACKGROUND: Plasma-based cell-free DNA is an attractive biospecimen for assessing somatic mutations due to minimally-invasive real-time sampling. However, next generation sequencing (NGS) of cell-free DNA (cfDNA) may not be appropriate for all patients with advanced prostate cancer (PC). METHODS: Blood was obtained from advanced PC patients for plasma-based sequencing. UW-OncoPlex, a ∼2 Mb multi-gene NGS panel performed in the CLIA/CAP environment, was optimized for detecting cfDNA mutations. Tumor tissue and germline samples were sequenced for comparative analyses. Multivariate logistic regression was performed to determine the clinical characteristic associated with the successful detection of somatic cfDNA alterations (ie detection of at least one clearly somatic PC mutation). RESULTS: Plasma for cfDNA sequencing was obtained from 93 PC patients along with tumor tissue (N = 67) and germline (N = 93) controls. We included data from 76 patients (72 prostate adenocarcinoma; 4 variant histology PC) in the analysis. Somatic DNA aberrations were detected in 34 cfDNA samples from patients with prostate adenocarcinoma. High PSA level, high tumor volume, and castration-resistance were significantly associated with successful detection of somatic cfDNA alterations. Among samples with somatic mutations detected, the cfDNA assay detected 93/102 (91%) alterations found in tumor tissue, yielding a clustering-corrected sensitivity of 92% (95% confidence interval 88-97%). All germline pathogenic variants present in lymphocyte DNA were also detected in cfDNA (N = 12). Somatic mutations from cfDNA were detected in 30/33 (93%) instances when PSA was >10 ng/mL. CONCLUSIONS: Disease burden, including a PSA >10 ng/mL, is strongly associated with detecting somatic mutations from cfDNA specimens.

Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer.

BACKGROUND: Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. METHODS: We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. RESULTS: A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). CONCLUSIONS: In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

Genome-wide mitochondrial DNA sequence variations and lower expression of OXPHOS genes predict mitochondrial dysfunction in oral cancer tissue.

Several studies reported that mtDNA mutations may play important roles in carcinogenesis although the mechanism is not clear yet. Most of the studies compared mtDNA sequences in a tumor with those in normal tissues from different individuals ignoring inter-individual variations. In this study, 271 SNPs, 7 novel SNPs (or SNVs), and 15 somatic mutations were detected in mtDNA of 8 oral cancer tissues with respect to reference (rCRS) and adjacent normal tissues, respectively, using Ion PGM next generation sequencing method. Most of the sequence variations (76 SNPs and 1 somatic) are present in D-loop region followed by CyB (36 SNPs), ATP6 (24 SNPs), ND5 (17 SNPs and 5 somatic), ND4 (18 coding and 2 somatic) and other non-coding and coding DNA sequences. A total of 53 and 8 non-synonymous SNPs and somatic mutations, respectively, were detected in tumor tissues and some of these variations may have deleterious effects on the protein function as predicted by bioinformatic analysis. Moreover, significantly low mtDNA contents and expression of several mitochondrial genes in tumor compared to adjacent normal tissues may have also affected mitochondrial functions. Taken together, this study suggests that mtDNA mutations as well as low expression of mtDNA coded genes may play important roles in tumor growth. Although the sample size is low, an important aspect of the study is the use of adjacent control tissues to find out somatic mutations and a change in the expression of mitochondrial genes, to rule out inter-individual and inter-tissue variations which are important issues in the study of mitochondrial genomics.

Genetic variations at microRNA and processing genes and risk of oral cancer.

Genetic variations at microRNA and microRNA processing genes are known to confer risk of cancer in different populations. Here, we studied variations at eight microRNA (miRNA) and four miRNA processing genes in 452 controls and 451 oral cancer patients by TaqMan genotyping assays. Variant allele-containing genotypes at mir-196a2 and variant allele homozygous genotype at Ran increased the risk of cancer significantly [adjusted odds ratio (OR) (95% confidence interval (CI)) = 1.3 (1-1.7) and 2.3 (1.1-4.6), respectively]. Conversely, variant allele-containing genotypes at mir-34b and variant allele homozygous genotype at Gemin3 reduced the risk of cancer significantly [adjusted OR (95% CI) = 0.7 (0.5-0.9) and 0.6 (0.4-1), respectively]. Cumulative risk was also increased by three times with increase in the number of risk alleles at these four loci. In tobacco stratified analysis, variant allele homozygous genotypes at mir-29a and Ran increased [adjusted OR (95% CI) = 1.5 (1-2.3) and 3 (1.1-8.4) respectively], while variant allele-containing genotypes at mir-34b decreased [adjusted OR (95% CI) = 0.6 (0.4-0.9)] the risk of cancer significantly. Thus, genetic variation at miRNA and processing genes altered the risk of oral cancer in this population thereby corroborating studies in other populations. However, it is necessary to validate this result in different Indian sub populations with larger sample sizes and examine the effect of these variations in tumour tissues to explain the mechanism of risk alteration.

Association between risk of oral precancer and genetic variations in microRNA and related processing genes.

BACKGROUND: MicroRNAs have been implicated in cancer but studies on their role in precancer, such as leukoplakia, are limited. Sequence variations at eight miRNA and four miRNA processing genes were studied in 452 healthy controls and 299 leukoplakia patients to estimate risk of disease. RESULTS: Genotyping by TaqMan assay followed by statistical analyses showed that variant genotypes at Gemin3 and mir-34b reduced risk of disease [OR = 0.5(0.3-0.9) and OR = 0.7(0.5-0.9) respectively] in overall patients as well as in smokers [OR = 0.58(0.3-1) and OR = 0.68(0.5-0.9) respectively]. Among chewers, only mir29a significantly increased risk of disease [OR = 1.8(1-3)]. Gene-environment interactions using MDR-pt program revealed that mir29a, mir34b, mir423 and Xpo5 modulated risk of disease (p < 0.002) which may be related to change in expression of these genes as observed by Real-Time PCR assays. But association between polymorphisms and gene expressions was not found in our sample set as well as in larger datasets from open access platforms like Genevar and 1000 Genome database. CONCLUSION: Variations in microRNAs and their processing genes modulated risk of precancer but further in-depth study is needed to understand mechanism of disease process.

Assessment of TROP2, CEACAM5 and DLL3 in metastatic prostate cancer: Expression landscape and molecular correlates.

Therapeutic approaches targeting proteins on the surface of cancer cells have emerged as an important strategy for precision oncology. To capitalize on the potential impact of drugs targeting surface proteins, detailed knowledge about the expression patterns of the target proteins in tumor tissues is required. In castration-resistant prostate cancer (CRPC), agents targeting prostate-specific membrane antigen (PSMA) have demonstrated clinical activity. However, PSMA expression is lost in a significant number of CRPC tumors. The identification of additional cell surface targets is necessary to develop new therapeutic approaches. Here, we performed a comprehensive analysis of the expression heterogeneity and co-expression patterns of trophoblast cell-surface antigen 2 (TROP2), delta-like ligand 3 (DLL3), and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in CRPC samples from a rapid autopsy cohort. We show that DLL3 and CEACAM5 exhibit the highest expression in neuroendocrine prostate cancer (NEPC), while TROP2 is expressed across different CRPC molecular subtypes, except for NEPC. We further demonstrated that AR alterations were associated with higher expression of PSMA and TROP2. Conversely, PSMA and TROP2 expression was lower in RB1-altered tumors. In addition to genomic alterations, we show a tight correlation between epigenetic states, particularly histone H3 lysine 27 methylation (H3K27me3) at the transcriptional start site and gene body of TACSTD2 (encoding TROP2), DLL3, and CEACAM5, and their respective protein expression in CRPC patient-derived xenografts. Collectively, these findings provide insights into patterns and determinants of expression of TROP2, DLL3, and CEACAM5 with implications for the clinical development of cell surface targeting agents in CRPC.

Bipolar androgen therapy plus olaparib in men with metastatic castration-resistant prostate cancer.

BACKGROUND: Bipolar androgen therapy (BAT) results in rapid fluctuation of testosterone (T) between near-castrate and supraphysiological levels and has shown promise in metastatic castration-resistant prostate cancer (mCRPC). Its clinical effects may be mediated through induction of DNA damage, and preclinical studies suggest synergy with PARP inhibitors. PATIENTS AND METHODS: This was a single-center, Phase II trial testing olaparib plus BAT (T cypionate/enanthate 400 mg every 28 days) with ongoing androgen deprivation. Planned recruitment was 30 subjects (equal proportions with/without homologous recombination repair [HRR] gene mutations) with mCRPC post abiraterone and/or enzalutamide. The primary objective was to determine PSA50 response (PSA decline ≥50% from baseline) rate at 12-weeks. The primary analysis utilized the entire (intent-to-treat [ITT]) cohort, with those dropping out early counted as non-responders. Secondary/exploratory analyses were in those treated beyond 12-weeks (response-evaluable cohort). RESULTS: Thirty-six patients enrolled and 6 discontinued prior to response assessment. In the ITT cohort, PSA50 response rate at 12-weeks was 11/36 (31%; 95% CI 17-48%), and 16/36 (44%, 95% CI 28-62%) had a PSA50 response at any time on-study. After a median follow-up of 19 months, the median clinical/radiographic progression-free survival in the ITT cohort was 13.0 months (95% CI 7-17). Clinical outcomes were similar regardless of HRR gene mutational status. CONCLUSIONS: BAT plus olaparib is associated with high response rates and long PFS. Clinical benefit was observed regardless of HRR gene mutational status.

Concurrent Targeting of HDAC and PI3K to Overcome Phenotypic Heterogeneity of Castration-resistant and Neuroendocrine Prostate Cancers.

Castration-resistant prostate cancer (CRPC) consists of multiple phenotypic subtypes including androgen receptor (AR)-active prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). Tumor cells with these phenotypes can coexist between metastases within a patient and within an individual tumor. Treatments that are effective across CRPC subtypes are currently lacking. Histone deacetylation is crucial for the regulation of chromatin structure and maintenance of cancer cell state and activation of the PI3K/AKT/mTOR signaling cascade is a tumor growth-promoting pathway. We therefore investigated combined targeting of histone deacetylase (HDAC) and PI3K using a rationally designed dual inhibitor, fimepinostat, in CRPC subtypes in vitro and in vivo. Dual HDAC1/2 and PI3K/AKT pathway inhibition by fimepinostat led to robust tumor growth inhibition in both ARPC and NEPC models including cell line- and patient-derived xenografts. HDAC1/2 inhibition combined with PI3K/AKT inhibition was more effective than targeting each pathway alone, producing growth inhibitory effects through cell-cycle inhibition and apoptosis. Molecular profiling revealed on-target effects of combined HDAC1/2 and PI3K/AKT inhibition independent of tumor phenotype. Fimepinostat therapy was also associated with the suppression of lineage transcription factors including AR in ARPC and Achaete-scute homolog 1 (ASCL1) in NEPC. Together, these results indicate that fimepinostat represents a novel therapeutic that may be effective against both ARPC and NEPC through CRPC subtype-dependent and -independent mechanisms. SIGNIFICANCE: CRPC is a heterogeneous disease constituting multiple phenotypic subtypes that often co-occur within tumors or across metastases in patients. Existing targeted therapies for CRPC do not take this into account. Here we show that fimepinostat, a dual HDAC1/2 and PI3K/AKT inhibitor investigated clinically in other cancer types but not prostate cancer, may overcome this heterogeneity by effectively inhibiting both ARPC and NEPC subtypes of CRPC.

Nucleosome Patterns in Circulating Tumor DNA Reveal Transcriptional Regulation of Advanced Prostate Cancer Phenotypes.

Advanced prostate cancers comprise distinct phenotypes, but tumor classification remains clinically challenging. Here, we harnessed circulating tumor DNA (ctDNA) to study tumor phenotypes by ascertaining nucleosome positioning patterns associated with transcription regulation. We sequenced plasma ctDNA whole genomes from patient-derived xenografts representing a spectrum of androgen receptor active (ARPC) and neuroendocrine (NEPC) prostate cancers. Nucleosome patterns associated with transcriptional activity were reflected in ctDNA at regions of genes, promoters, histone modifications, transcription factor binding, and accessible chromatin. We identified the activity of key phenotype-defining transcriptional regulators from ctDNA, including AR, ASCL1, HOXB13, HNF4G, and GATA2. To distinguish NEPC and ARPC in patient plasma samples, we developed prediction models that achieved accuracies of 97% for dominant phenotypes and 87% for mixed clinical phenotypes. Although phenotype classification is typically assessed by IHC or transcriptome profiling from tumor biopsies, we demonstrate that ctDNA provides comparable results with diagnostic advantages for precision oncology. SIGNIFICANCE: This study provides insights into the dynamics of nucleosome positioning and gene regulation associated with cancer phenotypes that can be ascertained from ctDNA. New methods for classification in phenotype mixtures extend the utility of ctDNA beyond assessments of somatic DNA alterations with important implications for molecular classification and precision oncology. This article is highlighted in the In This Issue feature, p. 517.

Co-occurring BRCA2/SPOP Mutations Predict Exceptional Poly (ADP-ribose) Polymerase Inhibitor Sensitivity in Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND AND OBJECTIVE: BRCA2 mutations in metastatic castration-resistant prostate cancer (mCRPC) confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. However, additional factors predicting PARP inhibitor efficacy in mCRPC are needed. Preclinical studies support a relationship between speckle-type POZ protein (SPOP) inactivation and PARP inhibitor sensitivity. We hypothesized that SPOP mutations may predict enhanced PARP inhibitor response in BRCA2-altered mCRPC. METHODS: We conducted a multicenter retrospective study involving 13 sites. We identified 131 patients with BRCA2-altered mCRPC treated with PARP inhibitors, 14 of which also carried concurrent SPOP mutations. The primary efficacy endpoint was prostate-specific antigen (PSA) response rate (≥50% PSA decline). The secondary endpoints were biochemical progression-free survival (PSA-PFS), clinical/radiographic progression-free survival (PFS), and overall survival (OS). These were compared by multivariable Cox proportional hazard models adjusting for age, tumor stage, baseline PSA level, Gleason sum, prior therapies, BRCA2 alteration types, and co-occurring mutations. KEY FINDINGS AND LIMITATIONS: Baseline characteristics were similar between groups. PSA responses were observed in 60% (70/117) of patients with BRCA2mut/SPOPwt disease and in 86% (12/14) of patients with BRCA2mut/SPOPmut disease (p = 0.06). The median time on PARP inhibitor treatment was 24.0 mo (95% confidence interval [CI] 19.2 mo to not reached) in this group versus 8.0 mo (95% CI 6.1-10.9 mo) in patients with BRCA2 mutation alone (p = 0.05). In an unadjusted analysis, patients with BRCA2mut/SPOPmut disease experienced longer PSA-PFS (hazard ratio [HR] 0.33 [95% CI 0.15-0.72], p = 0.005) and clinical/radiographic PFS (HR 0.4 [95% CI 0.18-0.86], p = 0.02), and numerically longer OS (HR 0.4 [95% CI 0.15-1.12], p = 0.08). In a multivariable analysis including histology, Gleason sum, prior taxane, prior androgen receptor pathway inhibitor, stage, PSA, BRCA2 alteration characteristics, and other co-mutations, patients with BRCA2mut/SPOPmut disease experienced longer PSA-PFS (HR 0.16 [95% CI 0.05-0.47], adjusted p = 0.001), clinical/radiographic PFS (HR 0.28 [95% CI 0.1-0.81], adjusted p = 0.019), and OS (HR 0.19 [95% CI 0.05-0.69], adjusted p = 0.012). In a separate cohort of patients not treated with a PARP inhibitor, there was no difference in OS between patients with BRCA2mut/SPOPmut versus BRCA2mut/SPOPwt disease (HR 0.97 [95% CI 0.40-2.4], p = 0.94). In a genomic signature analysis, Catalog of Somatic Mutations in Cancer (COSMIC) SBS3 scores predictive of homologous recombination repair (HRR) defects were higher for BRCA2mut/SPOPmut than for BRCA2mut/SPOPwt disease (p = 0.04). This was a retrospective study, and additional prospective validation cohorts are needed. CONCLUSIONS AND CLINICAL IMPLICATIONS: In this retrospective analysis, PARP inhibitors appeared more effective in patients with BRCA2mut/SPOPmut than in patients with BRCA2mut/SPOPwt mCRPC. This may be related to an increase in HRR defects in coaltered disease. PATIENT SUMMARY: In this study, we demonstrate that co-alteration of both BRCA2 and SPOP predicts superior clinical outcomes to treatment with poly (ADP-ribose) polymerase (PARP) inhibitors than BRCA2 alteration without SPOP mutation.

ASSESSMENT OF CELL SURFACE TARGETS IN METASTATIC PROSTATE CANCER: EXPRESSION LANDSCAPE AND MOLECULAR CORRELATES.

Therapeutic approaches targeting proteins on the surface of cancer cells have emerged as an important strategy for precision oncology. To fully capitalize on the potential impact of drugs targeting surface proteins, detailed knowledge about the expression patterns of the target proteins in tumor tissues is required. In castration-resistant prostate cancer (CRPC), agents targeting prostate-specific membrane antigen (PSMA) have demonstrated clinical activity. However, PSMA expression is lost in a significant number of CRPC tumors, and the identification of additional cell surface targets is necessary in order to develop new therapeutic approaches. Here, we performed a comprehensive analysis of the expression and co-expression patterns of trophoblast cell-surface antigen 2 (TROP2), delta-like ligand 3 (DLL3), and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in CRPC samples from a rapid autopsy cohort. We show that DLL3 and CEACAM5 exhibit the highest expression in neuroendocrine prostate cancer (NEPC), while TROP2 is expressed across different CRPC molecular subtypes, except for NEPC. We observed variable intra-tumoral and inter-tumoral heterogeneity and no dominant metastatic site predilections for TROP2, DLL3, and CEACAM5. We further show that AR amplifications were associated with higher expression of PSMA and TROP2 but lower DLL3 and CEACAM5 levels. Conversely, PSMA and TROP2 expression was lower in RB1-altered tumors. In addition to genomic alterations, we demonstrate a tight correlation between epigenetic states, particularly histone H3 lysine 27 methylation (H3K27me3) at the transcriptional start site and gene body of TACSTD2 (encoding TROP2), DLL3, and CEACAM5, and their respective protein expression in CRPC patient-derived xenografts. Collectively, these findings provide novel insights into the patterns and determinants of expression of TROP2, DLL3, and CEACAM5 with important implications for the clinical development of cell surface targeting agents in CRPC.

Differential haplotype amplification leads to misgenotyping of heterozygote as homozygote when using single nucleotide mismatch primer.

Mismatches at the 3'end of /or within a primer are reported to affect the efficiency of PCR and cause allele drop. Here, we report preferential amplification of one haplotype and misgenotyping, when double heterozygotes at NAT1 (rs1057126 and rs15561) were genotyped by sequencing and PCR-RFLP methods using mismatch reverse primers located next to the target SNP. Detailed study revealed highest (100%) and lowest (0%) misgenotyping when the mismatch was at the 3rd and 15th nucleotide positions from 3' end of the primer, respectively. But, the same primers, without any mismatch genotyped heterozygotes correctly. Homozygotes can always be detected correctly irrespective of mismatch position in the primer. Similar results were observed for two SNPs (rs12947788 and rs 12951053) at TP53. Using mismatch NAT1 reverse primers, located three nucleotides away from the target SNP, both TaqMan and sequencing methods showed preferential synthesis of one haplotype strand and misgenotyping in heterozygotes, respectively. So, mismatch primer, located next to target SNP, should be avoided to genotype heterozygotes, since, PCR and sequencing based genotyping methods may lead the investigators to report faulty allelic and genotypic frequencies. This study mimics a situation when an unknown variation is present in the primer-binding sites of both chromosomes.

A framework for clinical cancer subtyping from nucleosome profiling of cell-free DNA.

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we develop Griffin, a framework for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing data. Griffin employs a GC correction procedure tailored to variable cfDNA fragment sizes, which generates a better representation of chromatin accessibility and improves the accuracy of cancer detection and tumor subtype classification. We demonstrate estrogen receptor subtyping from cfDNA in metastatic breast cancer. We predict estrogen receptor subtype in 139 patients with at least 5% detectable circulating tumor DNA with an area under the receive operator characteristic curve (AUC) of 0.89 and validate performance in independent cohorts (AUC = 0.96). In summary, Griffin is a framework for accurate tumor subtyping and can be generalizable to other cancer types for precision oncology applications.

Concordance of DNA Repair Gene Mutations in Paired Primary Prostate Cancer Samples and Metastatic Tissue or Cell-Free DNA.

IMPORTANCE: DNA damage repair (DDR) gene mutations represent actionable alterations that can guide precision medicine strategies for advanced prostate cancer. However, acquisition of contemporary tissue samples for molecular testing can be a barrier to deploying precision medicine approaches. We hypothesized that most DDR alterations represent truncal events in prostate cancer and that primary tissue would faithfully reflect mutations found in cell-free circulating tumor DNA (ctDNA) and/or metastatic tissue. OBJECTIVE: To assess concordance in DDR gene alterations between primary prostate cancer and metastases or ctDNA specimens. DESIGN, SETTING, AND PARTICIPANTS: Patients were included if a DDR pathway mutation was detected in metastatic tissue or ctDNA and primary tissue sequencing was available for comparison. Sequencing data from 3 cohorts were analyzed: (1) FoundationOne, (2) University of Washington clinical cases (University of Washington-OncoPlex or Stand Up to Cancer-Prostate Cancer Foundation International Dream Team sequencing pipelines), and (3) University of Washington rapid autopsy series. Only pathogenic somatic mutations were included, and more than 30 days between primary tumor tissue and ctDNA and/or metastatic tissue acquisition was required. Clonal hematopoiesis of indeterminate potential (CHIP) and germline events were adjudicated by an expert molecular pathologist and excluded. MAIN OUTCOMES AND MEASURES: The DDR gene alterations detected in primary prostate tissue matched with metastatic tissue and/or ctDNA findings. RESULTS: A total of 72 men with known DDR alterations were included in the analysis, and primary samples with paired ctDNA and/or metastatic tissue were sequenced. After excluding patients with ctDNA where only CHIP and/or germline events (n = 21) were observed, 51 patients remained and were included in the final analysis. The median (range) time from acquisition of primary tissue to acquisition of ctDNA or tumor tissue was 55 (5-193) months. Concordance in DDR gene mutation status across samples was 84% (95% CI, 71%-92%). Rates of concordance between metastatic-primary and ctDNA-primary pairs were similar when patients with CHIP events were excluded. Multiclonal BRCA2 reversion mutations associated with resistance to PARP inhibitors and platinum chemotherapy were detected in ctDNA from 2 patients. CONCLUSIONS AND RELEVANCE: In this genetic association study of 3 patient cohorts, primary prostate tissue accurately reflected the mutational status of actionable DDR genes in metastatic tissue, consistent with DDR alterations being truncal in most patients. After excluding likely CHIP events, ctDNA profiling accurately captured these DDR mutations while also detecting reversion alterations that may suggest resistance mechanisms.

Regulation of CEACAM5 and Therapeutic Efficacy of an Anti-CEACAM5-SN38 Antibody-drug Conjugate in Neuroendocrine Prostate Cancer.

PURPOSE: Neuroendocrine prostate cancer (NEPC) is an aggressive form of castration-resistant prostate cancer (CRPC) for which effective therapies are lacking. We previously identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a promising NEPC cell surface antigen. Here we investigated the scope of CEACAM5 expression in end-stage prostate cancer, the basis for CEACAM5 enrichment in NEPC, and the therapeutic potential of the CEACAM5 antibody-drug conjugate labetuzumab govitecan in prostate cancer. EXPERIMENTAL DESIGN: The expression of CEACAM5 and other clinically relevant antigens was characterized by multiplex immunofluorescence of a tissue microarray comprising metastatic tumors from 34 lethal metastatic CRPC (mCRPC) cases. A genetically defined neuroendocrine transdifferentiation assay of prostate cancer was developed to evaluate mechanisms of CEACAM5 regulation in NEPC. The specificity and efficacy of labetuzumab govitecan was determined in CEACAM5+ prostate cancer cell lines and patient-derived xenografts models. RESULTS: CEACAM5 expression was enriched in NEPC compared with other mCRPC subtypes and minimally overlapped with prostate-specific membrane antigen, prostate stem cell antigen, and trophoblast cell surface antigen 2 expression. We focused on a correlation between the expression of the pioneer transcription factor ASCL1 and CEACAM5 to determine that ASCL1 can drive neuroendocrine reprogramming of prostate cancer which is associated with increased chromatin accessibility of the CEACAM5 core promoter and CEACAM5 expression. Labetuzumab govitecan induced DNA damage in CEACAM5+ prostate cancer cell lines and marked antitumor responses in CEACAM5+ CRPC xenograft models including chemotherapy-resistant NEPC. CONCLUSIONS: Our findings provide insights into the scope and regulation of CEACAM5 expression in prostate cancer and strong support for clinical studies of labetuzumab govitecan for NEPC.

Selective androgen receptor modulators activate the canonical prostate cancer androgen receptor program and repress cancer growth.

Prostate cancer (PC) is driven by androgen receptor (AR) activity, a master regulator of prostate development and homeostasis. Frontline therapies for metastatic PC deprive the AR of the activating ligands testosterone (T) and dihydrotestosterone (DHT) by limiting their biosynthesis or blocking AR binding. Notably, AR signaling is dichotomous, inducing growth at lower activity levels, while suppressing growth at higher levels. Recent clinical studies have exploited this effect by administration of supraphysiological concentrations of T, resulting in clinical responses and improvements in quality of life. However, the use of T as a therapeutic agent in oncology is limited by poor drug-like properties as well as rapid and variable metabolism. Here, we investigated the antitumor effects of selective AR modulators (SARMs), which are small-molecule nonsteroidal AR agonists developed to treat muscle wasting and cachexia. Several orally administered SARMs activated the AR program in PC models. AR cistromes regulated by steroidal androgens and SARMs were superimposable. Coregulatory proteins including HOXB13 and GRHL2 comprised AR complexes assembled by both androgens and SARMs. At bioavailable concentrations, SARMs repressed MYC oncoprotein expression and inhibited the growth of castration-sensitive and castration-resistant PC in vitro and in vivo. These results support further clinical investigation of SARMs for treating advanced PC.

Genomic attributes of homology-directed DNA repair deficiency in metastatic prostate cancer.

Cancers with homology-directed DNA repair (HRR) deficiency exhibit high response rates to poly(ADP-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy. Though mutations disrupting BRCA1 and BRCA2 associate with HRR deficiency (HRRd), patterns of genomic aberrations and mutation signatures may be more sensitive and specific indicators of compromised repair. Here, we evaluated whole-exome sequences from 418 metastatic prostate cancers (mPCs) and determined that one-fifth exhibited genomic characteristics of HRRd that included Catalogue Of Somatic Mutations In Cancer mutation signature 3. Notably, a substantial fraction of tumors with genomic features of HRRd lacked biallelic loss of a core HRR-associated gene, such as BRCA2. In this subset, HRRd associated with loss of chromodomain helicase DNA binding protein 1 but not with mutations in serine-protein kinase ATM, cyclin dependent kinase 12, or checkpoint kinase 2. HRRd genomic status was strongly correlated with responses to PARPi and platinum chemotherapy, a finding that supports evaluating biomarkers reflecting functional HRRd for treatment allocation.