Antagonism of inhibitors of apoptosis proteins reveals a novel, immune response-based therapeutic approach for T-cell lymphoma.

Tolinapant (ASTX660) is a potent, nonpeptidomimetic antagonist of cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) and X-linked IAP, which is currently being evaluated in a phase 2 study in T-cell lymphoma (TCL) patients. Tolinapant has demonstrated evidence of single-agent clinical activity in relapsed/refractory peripheral TCL and cutaneous TCL. To investigate the mechanism of action underlying the single-agent activity observed in the clinic, we have used a comprehensive translational approach integrating in vitro and in vivo models of TCL confirmed by data from human tumor biopsies. Here, we show that tolinapant acts as an efficacious immunomodulatory molecule capable of inducing complete tumor regression in a syngeneic model of TCL exclusively in the presence of an intact immune system. These findings were confirmed in samples from our ongoing clinical study showing that tolinapant treatment can induce changes in gene expression and cytokine profile consistent with immune modulation. Mechanistically, we show that tolinapant can activate both the adaptive and the innate arms of the immune system through the induction of immunogenic forms of cell death. In summary, we describe a novel role for IAP antagonists as immunomodulatory molecules capable of promoting a robust antitumor immune response in TCL.

ASTX029, a Novel Dual-mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK.

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I-II clinical trial in patients with advanced solid tumors (NCT03520075).

First-in-Human Study of AT13148, a Dual ROCK-AKT Inhibitor in Patients with Solid Tumors.

PURPOSE: AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity. PATIENTS AND METHODS: The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon-Wed-Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3ß levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies. RESULTS: Fifty-one patients were treated on study. The safety of 5-300 mg of AT13148 was studied. Further, the doses of 120-180-240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean C max and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies. CONCLUSIONS: AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer.

A Phase I Study of ASTX660, an Antagonist of Inhibitors of Apoptosis Proteins, in Adults with Advanced Cancers or Lymphoma.

PURPOSE: This first-in-human, phase I study evaluated ASTX660, an oral, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins in patients with advanced solid tumors or lymphoma. PATIENTS AND METHODS: ASTX660 was administered orally once daily on a 7-day-on/7-day-off schedule in a 28-day cycle. Dose escalation followed a standard 3+3 design to determine the MTD and recommended phase II dose (RP2D). Dose expansion was conducted at the RP2D. RESULTS: Forty-five patients received ASTX660 (range 15-270 mg/day). Dose-limiting toxicity of grade 3 increased lipase with or without increased amylase occurred in 3 patients at 270 mg/day and 1 patient at 210 mg/day. The MTD was determined to be 210 mg/day and the RP2D 180 mg/day. Common treatment-related adverse events included fatigue (33%), vomiting (31%), and nausea (27%). Grade ≥3 treatment-related adverse events occurred in 7 patients, most commonly anemia (13%), increased lipase (11%), and lymphopenia (9%). ASTX660 was rapidly absorbed, with maximum concentration achieved at approximately 0.5-1.0 hour. An approximately 2-fold accumulation in AUC exposures was observed on day 7 versus 1. ASTX660 suppressed cellular inhibitor of apoptosis protein-1 in peripheral blood mononuclear cells, which was maintained into the second cycle beyond the off-therapy week at the 180-mg/day RP2D and above. Clinical activity was seen in a patient with cutaneous T-cell lymphoma. CONCLUSIONS: ASTX660 demonstrated a manageable safety profile and exhibited evidence of pharmacodynamic and preliminary clinical activity at the 180-mg/day RP2D. The phase II part of the study is ongoing.

Pharmacodynamic and Clinical Results from a Phase I/II Study of the HSP90 Inhibitor Onalespib in Combination with Abiraterone Acetate in Prostate Cancer.

PURPOSE: Onalespib is a potent, fragment-derived second-generation HSP90 inhibitor with preclinical activity in castration-resistant prostate cancer (CPRC) models. This phase I/II trial evaluated onalespib in combination with abiraterone acetate (AA) and either prednisone or prednisolone (P) in men with CRPC progressing on AA/P. PATIENTS AND METHODS: Patients with progressing CRPC were randomly assigned to receive 1 of 2 regimens of onalespib combined with AA/P. Onalespib was administered as intravenous infusion starting at 220 mg/m2 once weekly for 3 of 4 weeks (regimen 1); or at 120 mg/m2 on day 1 and day 2 weekly for 3 of 4 weeks (regimen 2). Primary endpoints were response rate and safety. Secondary endpoints included evaluation of androgen receptor (AR) depletion in circulating tumor cells (CTC) and in fresh tumor tissue biopsies. RESULTS: Forty-eight patients were treated with onalespib in combination with AA/P. The most common ≥grade 3 toxicities related to onalespib included diarrhea (21%) and fatigue (13%). Diarrhea was dose limiting at 260 and 160 mg/m2 for regimens 1 and 2, respectively. Transient decreases in CTC counts and AR expression in CTC were observed in both regimens. HSP72 was significantly upregulated following onalespib treatment, but only a modest decrease in AR and GR was shown in paired pre- and posttreatment tumor biopsy samples. No patients showed an objective or PSA response. CONCLUSIONS: Onalespib in combination with AA/P showed mild evidence of some biological effect; however, this effect did not translate into clinical activity, hence further exploration of this combination was not justified.

Immunomodulatory Properties of DNA Hypomethylating Agents: Selecting the Optimal Epigenetic Partner for Cancer Immunotherapy.

DNA hypomethylating agents (DHAs) play a well-acknowledged role in potentiating the immunogenicity and the immune recognition of neoplastic cells. This immunomodulatory activity of DHAs is linked to their ability to induce or to up-regulate on neoplastic cells the expression of a variety of immune molecules that play a crucial role in host-tumor immune interactions. To further investigate the clinical potential of diverse epigenetic compounds when combined with immunotherapeutic strategies, we have now compared the tumor immunomodulatory properties of the first generation DHAs, azacytidine (AZA) and decitabine (DAC) and of the next generation DHA, guadecitabine. To this end, human melanoma and hematological cancer cells were treated in vitro with 1 µM guadecitabine, DAC or AZA and then studied by molecular and flow cytometry analyses for changes in their baseline expression of selected immune molecules involved in different mechanism(s) of immune recognition. Results demonstrated a stronger DNA hypomethylating activity of guadecitabine and DAC, compared to AZA that associated with stronger immunomodulatory activities. Indeed, the mRNA expression of cancer testis antigens, immune-checkpoint blocking molecules, immunostimulatory cytokines, involved in NK and T cell signaling and recruiting, and of genes involved in interferon pathway was higher after guadecitabine and DAC compared to AZA treatment. Moreover, a stronger up-regulation of the constitutive expression of HLA class I antigens and of Intercellular Adhesion Molecule-1 was observed with guadecitabine and DAC compared to AZA. Guadecitabine and DAC seem to represent the optimal combination partners to improve the therapeutic efficacy of immunotherapeutic agents in combination/sequencing clinical studies.

Docetaxel Treatment in PTEN- and ERG-aberrant Metastatic Prostate Cancers.

BACKGROUND: Loss of PTEN is a common genomic aberration in castration-resistant prostate cancer (CRPC) and is frequently concurrent with ERG rearrangements, causing resistance to next-generation hormonal treatment (NGHT) including abiraterone. The relationship between PTEN loss and docetaxel sensitivity remains uncertain. OBJECTIVE: To study the antitumor activity of docetaxel in metastatic CRPC in relation to PTEN and ERG aberrations. DESIGN SETTING AND PARTICIPANTS: Single-centre, retrospective analysis of PTEN loss and ERG expression using a previously described immunohistochemistry (IHC) binary classification system. Patients received docetaxel between January 1, 2006 and July 31, 2016. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Response correlations were analyzed using Pearson's χ2 tests and independent-sample t tests. Overall (OS) and progression-free survival (PFS) were analyzed using univariate and multivariate (MVA) Cox regression and Kaplan-Meier methods. RESULTS AND LIMITATIONS: Overall, 215 patients were eligible. Established metastatic CRPC prognostic factors were well balanced between PTEN loss (39%) and normal patients (61%). PTEN loss was associated with shorter median OS (25.4 vs 34.7 mo; hazard ratio [HR] 1.66, 95% confidence interval [CI] 1.18-2.13; p = 0.001). There were no differences in median PFS (8.0 vs 9.1 mo; univariate HR 1.20, 95% CI 0.86-1.68; p = 0.28) and PSA response (53.4% vs 50.6%; p = 0.74). PTEN loss was an independent prognostics factor in MVA. ERG status was available for 100 patients. ERG positivity was not associated with OS or PFS. Limitations include the retrospective nature and the single-centre analysis. CONCLUSIONS: Our findings suggest that metastatic CRPC with PTEN loss might benefit more from docetaxel than from NGHT. PATIENT SUMMARY: In this study we found that metastatic prostate cancer with loss of the PTEN switch may benefit more from docetaxel than from abiraterone.

Gene Copy Number Estimation from Targeted Next-Generation Sequencing of Prostate Cancer Biopsies: Analytic Validation and Clinical Qualification.

Purpose: Precise detection of copy number aberrations (CNA) from tumor biopsies is critically important to the treatment of metastatic prostate cancer. The use of targeted panel next-generation sequencing (NGS) is inexpensive, high throughput, and easily feasible, allowing single-nucleotide variant calls, but CNA estimation from this remains challenging.Experimental Design: We evaluated CNVkit for CNA identification from amplicon-based targeted NGS in a cohort of 110 fresh castration-resistant prostate cancer biopsies and used capture-based whole-exome sequencing (WES), array comparative genomic hybridization (aCGH), and FISH to explore the viability of this approach.Results: We showed that this method produced highly reproducible CNA results (r = 0.92), with the use of pooled germline DNA as a coverage reference supporting precise CNA estimation. CNA estimates from targeted NGS were comparable with WES (r = 0.86) and aCGH (r = 0.7); for key selected genes (BRCA2, MYC, PIK3CA, PTEN, and RB1), CNA estimation correlated well with WES (r = 0.91) and aCGH (r = 0.84) results. The frequency of CNAs in our population was comparable with that previously described (i.e., deep deletions: BRCA2 4.5%; RB1 8.2%; PTEN 15.5%; amplification: AR 45.5%; gain: MYC 31.8%). We also showed, utilizing FISH, that CNA estimation can be impacted by intratumor heterogeneity and demonstrated that tumor microdissection allows NGS to provide more precise CNA estimates.Conclusions: Targeted NGS and CNVkit-based analyses provide a robust, precise, high-throughput, and cost-effective method for CNA estimation for the delivery of more precise patient care. Clin Cancer Res; 23(20); 6070-7. ©2017 AACR.

Phenotypic diversity of circulating tumour cells in patients with metastatic castration-resistant prostate cancer.

OBJECTIVES: To use a non-biased assay for circulating tumour cells (CTCs) in patients with prostate cancer (PCa) in order to identify non-traditional CTC phenotypes potentially excluded by conventional detection methods that are reliant on antigen- and/or size-based enrichment. PATIENTS AND METHODS: A total of 41 patients with metastatic castration-resistant PCa (mCRPC) and 20 healthy volunteers were analysed on the Epic CTC platform, via high-throughput imaging of DAPI expression and CD45/cytokeratin (CK) immunofluorescence (IF) on all circulating nucleated cells plated on glass slides. To confirm the PCa origin of CTCs, IF was used for androgen receptor (AR) expression and fluorescence in situ hybridization was used for PTEN and ERG assessment. RESULTS: Traditional CTCs (CD45- /CK+ /morphologically distinct) were identified in all patients with mCRPC and we also identified CTC clusters and non-traditional CTCs in patients with mCRPC, including CK- and apoptotic CTCs. Small CTCs (≤white blood cell size) were identified in 98% of patients with mCRPC. Total, traditional and non-traditional CTCs were significantly increased in patients who were deceased vs alive after 18 months; however, only non-traditional CTCs were associated with overall survival. Traditional and total CTC counts according to the Epic platform in the mCRPC cohort were also significantly correlated with CTC counts according to the CellSearch system. CONCLUSIONS: Heterogeneous non-traditional CTC populations are frequent in mCRPC and may provide additional prognostic or predictive information.

Drug discovery in advanced prostate cancer: translating biology into therapy.

Castration-resistant prostate cancer (CRPC) is associated with a poor prognosis and poses considerable therapeutic challenges. Recent genetic and technological advances have provided insights into prostate cancer biology and have enabled the identification of novel drug targets and potent molecularly targeted therapeutics for this disease. In this article, we review recent advances in prostate cancer target identification for drug discovery and discuss their promise and associated challenges. We review the evolving therapeutic landscape of CRPC and discuss issues associated with precision medicine as well as challenges encountered with immunotherapy for this disease. Finally, we envision the future management of CRPC, highlighting the use of circulating biomarkers and modern clinical trial designs.

Clinical development of new drug-radiotherapy combinations.

In countries with the best cancer outcomes, approximately 60% of patients receive radiotherapy as part of their treatment, which is one of the most cost-effective cancer treatments. Notably, around 40% of cancer cures include the use of radiotherapy, either as a single modality or combined with other treatments. Radiotherapy can provide enormous benefit to patients with cancer. In the past decade, significant technical advances, such as image-guided radiotherapy, intensity-modulated radiotherapy, stereotactic radiotherapy, and proton therapy enable higher doses of radiotherapy to be delivered to the tumour with significantly lower doses to normal surrounding tissues. However, apart from the combination of traditional cytotoxic chemotherapy with radiotherapy, little progress has been made in identifying and defining optimal targeted therapy and radiotherapy combinations to improve the efficacy of cancer treatment. The National Cancer Research Institute Clinical and Translational Radiotherapy Research Working Group (CTRad) formed a Joint Working Group with representatives from academia, industry, patient groups and regulatory bodies to address this lack of progress and to publish recommendations for future clinical research. Herein, we highlight the Working Group's consensus recommendations to increase the number of novel drugs being successfully registered in combination with radiotherapy to improve clinical outcomes for patients with cancer.

Castration-Resistant Prostate Cancer Tissue Acquisition From Bone Metastases for Molecular Analyses.

BACKGROUND: The urgent need for castration-resistant prostate cancer molecular characterization to guide treatment has been constrained by the disease's predilection to metastasize primarily to bone. We hypothesized that the use of clinical and imaging criteria could maximize tissue acquisition from bone marrow biopsies (BMBs). We aimed to develop a score for the selection of patients undergoing BMB. MATERIALS AND METHODS: A total of 115 BMBs were performed in 101 patients: 57 were included in a derivation set and 58 were used as the validation set. The clinical and laboratory data and prebiopsy computed tomography parameters (Hounsfield units [HUs]) were determined. A score for the prediction of biopsy positivity was developed from logistic regression analysis of the derivation set and tested in the validation set. RESULTS: Of the 115 biopsy specimens, 75 (62.5%) were positive; 35 (61.4%) in the test set and 40 (69%) in the validation set. On univariable analysis, hemoglobin (P = .019), lactate dehydrogenase (P = .003), prostate-specific antigen (P = .005), and mean HUs (P = .004) were selected. A score based on the LDH level (≥ 225 IU/L) and mean HUs (≥ 125) was developed in multivariate analysis and was associated with BMB positivity in the validation set (odds ratio, 5.1; 95% confidence interval, 1.9%-13.4%; P = .001). The area under the curve of the score was 0.79 in the test set and 0.77 in the validation set. CONCLUSION: BMB of the iliac crest is a feasible technique for obtaining tumor tissue for genomic analysis in patients with castration-resistant prostate cancer metastatic to the bone. A signature based on the mean HUs and LDH level can predict a positive yield with acceptable internal validity. Prospective studies of independent cohorts are needed to establish the external validity of the score.

Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells.

Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR.

Prostate-specific Antigen Decline After 4 Weeks of Treatment with Abiraterone Acetate and Overall Survival in Patients with Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: The availability of multiple new treatments for metastatic castration-resistant prostate cancer (mCRPC) mandates earlier treatment switches in the absence of a response. A decline in prostate-specific antigen (PSA) is widely used to monitor treatment response, but is not validated as an intermediate endpoint for overall survival (OS). OBJECTIVE: To evaluate the association between early PSA decline and OS following abiraterone acetate (AA) treatment. DESIGN, SETTING, AND PARTICIPANTS: We identified mCRPC patients treated with AA before or after docetaxel at the Royal Marsden NHS Foundation Trust between 2006 and 2014. Early PSA decline was defined as a 30% decrease in PSA at 4 wk relative to baseline, and early PSA rise as a 25% increase. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association with OS was analyzed using multivariate Cox regression and log-rank analyses. Spearman's rho correlation coefficient (r) was calculated to evaluate the association between PSA changes at 4 wk and 12 wk. RESULTS AND LIMITATIONS: There were 274 patients eligible for this analysis. A 30% PSA decline at 4 wk was associated with longer OS (25.8 vs 15.1 mo; hazard ratio [HR] 0.47, p<0.001), and a 25% PSA rise at 4 wk with shorter OS (15.1 vs 23.8 mo; HR 1.7, p=0.001) in both univariate and multivariable models. The percentage PSA decline at 4 wk was significantly correlated with the percentage PSA change at 12 wk (r=0.82; p<0.001). Patients achieving a 30% PSA decline at 4 wk were 11.7 times more likely to achieve a 50% PSA decrease at 12 wk (sensitivity 90.9%, specificity 79.4%). Limitations include the retrospective design of this analysis. CONCLUSIONS: Patients not achieving 30% PSA decline after 4 wk of AA have a lower likelihood of achieving PSA response at 12 wk and significantly inferior OS. Prospective multicentre validation studies are needed to confirm these findings. PATIENT SUMMARY: Prostate-specific antigen (PSA) is commonly used to evaluate response to treatment in metastatic castration-resistant prostate cancer. Expert recommendations discourage reliance on PSA changes earlier than 12 wk after treatment initiation. Our data suggest that early PSA changes are associated with survival in patients receiving abiraterone acetate.

Plasma AR and abiraterone-resistant prostate cancer.

Androgen receptor (AR) gene aberrations are rare in prostate cancer before primary hormone treatment but emerge with castration resistance. To determine AR gene status using a minimally invasive assay that could have broad clinical utility, we developed a targeted next-generation sequencing approach amenable to plasma DNA, covering all AR coding bases and genomic regions that are highly informative in prostate cancer. We sequenced 274 plasma samples from 97 castration-resistant prostate cancer patients treated with abiraterone at two institutions. We controlled for normal DNA in patients' circulation and detected a sufficiently high tumor DNA fraction to quantify AR copy number state in 217 samples (80 patients). Detection of AR copy number gain and point mutations in plasma were inversely correlated, supported further by the enrichment of nonsynonymous versus synonymous mutations in AR copy number normal as opposed to AR gain samples. Whereas AR copy number was unchanged from before treatment to progression and no mutant AR alleles showed signal for acquired gain, we observed emergence of T878A or L702H AR amino acid changes in 13% of tumors at progression on abiraterone. Patients with AR gain or T878A or L702H before abiraterone (45%) were 4.9 and 7.8 times less likely to have a ≥50 or ≥90% decline in prostate-specific antigen (PSA), respectively, and had a significantly worse overall [hazard ratio (HR), 7.33; 95% confidence interval (CI), 3.51 to 15.34; P = 1.3 × 10(-9)) and progression-free (HR, 3.73; 95% CI, 2.17 to 6.41; P = 5.6 × 10(-7)) survival. Evaluation of plasma AR by next-generation sequencing could identify cancers with primary resistance to abiraterone.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer.

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

PTEN loss in circulating tumour cells correlates with PTEN loss in fresh tumour tissue from castration-resistant prostate cancer patients.

BACKGROUND: PTEN gene loss occurs frequently in castration-resistant prostate cancer (CRPC) and may drive progression through activation of the PI3K/AKT pathway. Here, we developed a novel CTC-based assay to determine PTEN status and examined the correlation between PTEN status in CTCs and matched tumour tissue samples. METHODS: PTEN gene status in CTCs was evaluated on an enrichment-free platform (Epic Sciences) by fluorescence in situ hybridisation (FISH). PTEN status in archival and fresh tumour tissue was evaluated by FISH and immunohistochemistry. RESULTS: Peripheral blood was collected from 76 patients. Matched archival and fresh cancer tissue was available for 48 patients. PTEN gene status detected in CTCs was concordant with PTEN status in matched fresh tissues and archival tissue in 32 of 38 patients (84%) and 24 of 39 patients (62%), respectively. CTC counts were prognostic (continuous, P=0.001). PTEN loss in CTCs associated with worse survival in univariate analysis (HR 2.05; 95% CI 1.17-3.62; P=0.01) and with high lactate dehydrogenase (LDH) in metastatic CRPC patients. CONCLUSIONS: Our results illustrate the potential use of CTCs as a non-invasive, real-time liquid biopsy to determine PTEN gene status. The prognostic and predictive value of PTEN in CTCs warrants investigation in CRPC clinical trials of PI3K/AKT-targeted therapies.

Maximizing the Therapeutic Potential of HSP90 Inhibitors.

HSP90 is required for maintaining the stability and activity of a diverse group of client proteins, including protein kinases, transcription factors, and steroid hormone receptors involved in cell signaling, proliferation, survival, oncogenesis, and cancer progression. Inhibition of HSP90 alters the HSP90-client protein complex, leading to reduced activity, misfolding, ubiquitination, and, ultimately, proteasomal degradation of client proteins. HSP90 inhibitors have demonstrated significant antitumor activity in a wide variety of preclinical models, with evidence of selectivity for cancer versus normal cells. In the clinic, however, the efficacy of this class of therapeutic agents has been relatively limited to date, with promising responses mainly observed in breast and lung cancer, but no major activity seen in other tumor types. In addition, adverse events and some significant toxicities have been documented. Key to improving these clinical outcomes is a better understanding of the cellular consequences of inhibiting HSP90 that may underlie treatment response or resistance. This review considers the recent progress that has been made in the study of HSP90 and its inhibitors and highlights new opportunities to maximize their therapeutic potential.

Serial Next-Generation Sequencing of Circulating Cell-Free DNA Evaluating Tumor Clone Response To Molecularly Targeted Drug Administration.

PURPOSE: We evaluated whether next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA) could be used for patient selection and as a tumor clone response biomarker in patients with advanced cancers participating in early-phase clinical trials of targeted drugs. EXPERIMENTAL DESIGN: Plasma samples from patients with known tumor mutations who completed at least two courses of investigational targeted therapy were collected monthly, until disease progression. NGS was performed sequentially on the Ion Torrent PGM platform. RESULTS: cfDNA was extracted from 39 patients with various tumor types. Treatments administered targeted mainly the PI3K-AKT-mTOR pathway (n = 28) or MEK (n = 7). Overall, 159 plasma samples were sequenced with a mean sequencing coverage achieved of 1,685X across experiments. At trial initiation (C1D1), 23 of 39 (59%) patients had at least one mutation identified in cfDNA (mean 2, range 1-5). Out of the 44 mutations identified at C1D1, TP53, PIK3CA and KRAS were the top 3 mutated genes identified, with 18 (41%), 9 (20%), 8 (18%) different mutations, respectively. Out of these 23 patients, 13 received a targeted drug matching their tumor profile. For the 23 patients with cfDNA mutation at C1D1, the monitoring of mutation allele frequency (AF) in consecutive plasma samples during treatment with targeted drugs demonstrated potential treatment associated clonal responses. Longitudinal monitoring of cfDNA samples with multiple mutations indicated the presence of separate clones behaving discordantly. Molecular changes at cfDNA mutation level were associated with time to disease progression by RECIST criteria. CONCLUSIONS: Targeted NGS of cfDNA has potential clinical utility to monitor the delivery of targeted therapies.

Integrative clinical genomics of advanced prostate cancer.

Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%-60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, ß-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could impact treatment decisions for these affected individuals.