Robert B. Den

A Hormone–DNA Repair Circuit Governs the Response to Genotoxic Insult

UNLABELLED Alterations in DNA repair promote tumor development, but the impact on tumor progression is poorly understood. Here, discovery of a biochemical circuit linking hormone signaling to DNA repair and therapeutic resistance is reported. Findings show that androgen receptor (AR) activity is induced by DNA damage and promotes expression and activation of a gene expression program governing DNA repair. Subsequent investigation revealed that activated AR promotes resolution of double-strand breaks and resistance to DNA damage both in vitro and in vivo. Mechanistically, DNA-dependent protein kinase catalytic subunit (DNAPKcs) was identified as a key target of AR after damage, controlling AR-mediated DNA repair and cell survival after genotoxic insult. Finally, DNAPKcs was shown to potentiate AR function, consistent with a dual role in both DNA repair and transcriptional regulation. Combined, these studies identify the AR-DNAPKcs circuit as a major effector of DNA repair and therapeutic resistance and establish a new node for therapeutic intervention in advanced disease. SIGNIFICANCE The present study identifies for the fi rst time a positive feedback circuit linking hormone action to the DNA damage response and shows the significant impact of this process on tumor progression and therapeutic response. These provocative findings provide the foundation for development of novel nodes of therapeutic intervention for advanced disease.

RNA biomarkers associated with metastatic progression in prostate cancer: a multi-institutional high-throughput analysis of SChLAP1.

BACKGROUND Improved clinical predictors for disease progression are needed for localised prostate cancer, since only a subset of patients develop recurrent or refractory disease after first-line treatment. Therefore, we undertook an unbiased analysis to identify RNA biomarkers associated with metastatic progression after prostatectomy. METHODS Prostate cancer samples from patients treated with radical prostatectomy at three academic institutions were analysed for gene expression by a high-density Affymetrix GeneChip platform, encompassing more than 1 million genomic loci. In a discovery cohort, all protein-coding genes and known long non-coding RNAs were ranked by fold change in expression between tumours that subsequently metastasised versus those that did not. The top ranked gene was then validated for its prognostic value for metastatic progression in three additional independent cohorts. 95% of the gene expression assays were done in a Clinical Laboratory Improvements Amendments certified laboratory facility. All genes were assessed for their ability to predict metastatic progression by receiver-operating-curve area-under-the-curve analyses. Multivariate analyses were done for the primary endpoint of metastatic progression, with variables including Gleason score, preoperative prostate-specific antigen concentration, seminal vesicle invasion, surgical margin status, extracapsular extension, lymph node invasion, and expression of the highest ranked gene. FINDINGS 1008 patients were included in the study: 545 in the discovery cohort and 463 in the validation cohorts. The long non-coding RNA SChLAP1 was identified as the highest-ranked overexpressed gene in cancers with metastatic progression. Validation in three independent cohorts confirmed the prognostic value of SChLAP1 for metastatic progression. On multivariate modelling, SChLAP1 expression (high vs low) independently predicted metastasis within 10 years (odds ratio [OR] 2·45, 95% CI 1·70-3·53; p<0·0001). The only other variable that independently predicted metastasis within 10 years was Gleason score (8-10 vs 5-7; OR 2·14, 95% CI 1·77-2·58; p<0·0001). INTERPRETATION We identified and validated high SChLAP1 expression as significantly prognostic for metastatic disease progression of prostate cancer. Our findings suggest that further development of SChLAP1 as a potential biomarker, for treatment intensification in aggressive prostate cancer, warrants future study. FUNDING Prostate Cancer Foundation, National Institutes of Health, Department of Defense, Early Detection Research Network, Doris Duke Charitable Foundation, and Howard Hughes Medical Institute.

Daily image guidance with cone-beam computed tomography for head-and-neck cancer intensity-modulated radiotherapy: a prospective study.

PURPOSE To report on a prospective clinical trial of the use of daily kilovoltage cone-beam computed tomography (CBCT) to evaluate the interfraction and residual error motion of patients undergoing intensity-modulated radiotherapy for head-and-neck cancer. METHODS AND MATERIALS Patients were treated with intensity-modulated radiotherapy with an Elekta linear accelerator using a mounted CBCT scanner. CBCT was performed before every treatment, and translational (but not rotational) corrections were performed. At least once per week, a CBCT scan was obtained after intensity-modulated radiotherapy. Variations were measured in the medial-lateral, superoinferior, and anteroposterior dimensions, as well as in the rotation around these axes. RESULTS A total of 28 consecutive patients (1,013 CBCT scans) were studied. The average interfraction shift was 1.4 +/- 1.4, 1.7 +/- 1.9, and 1.8 +/- 2.1 mm in the medial-lateral, superoinferior, and anteroposterior dimensions, respectively. The corresponding average residual error shifts were 0.7 +/- 0.8, 0.9 +/- 0.9, and 0.9 +/- 0.9 mm. These data indicate that in the absence of daily CBCT image-guided radiotherapy, a clinical target volume to planning target volume margin of 3.9, 4.1, and 4.9 mm is needed in the medial-lateral, superoinferior, and anteroposterior dimensions, respectively. With daily CBCT, corresponding margins of 1.6, 2.5, and 1.9 mm should be acceptable. Subgroup analyses showed that larynx cancers and/or intratreatment weight loss indicate a need for slightly larger clinical target volume to planning target volume margins. CONCLUSION The results of our study have shown that image-guided radiotherapy using CBCT for head-and-neck cancer is effective. These data suggest it allows a reduction in the clinical target volume to planning target volume margins by about 50%, which could facilitate future studies of dose escalation and/or improved toxicity reduction. Caution is particularly warranted for cases in which the targets are mobile (e.g., the tongue).

Genomic classifier identifies men with adverse pathology after radical prostatectomy who benefit from adjuvant radiation therapy

PURPOSE The optimal timing of postoperative radiotherapy (RT) after radical prostatectomy (RP) is unclear. We hypothesized that a genomic classifier (GC) would provide prognostic and predictive insight into the development of clinical metastases in men receiving post-RP RT and inform decision making. PATIENTS AND METHODS GC scores were calculated from 188 patients with pT3 or margin-positive prostate cancer, who received post-RP RT at Thomas Jefferson University and Mayo Clinic between 1990 and 2009. The primary end point was clinical metastasis. Prognostic accuracy of the models was tested using the concordance index for censored data and decision curve analysis. Cox regression analysis tested the relationship between GC and metastasis. RESULTS The cumulative incidence of metastasis at 5 years after RT was 0%, 9%, and 29% for low, average, and high GC scores, respectively (P = .002). In multivariable analysis, GC and pre-RP prostate-specific antigen were independent predictors of metastasis (both P < .01). Within the low GC score (< 0.4), there were no differences in the cumulative incidence of metastasis comparing patients who received adjuvant or salvage RT (P = .79). However, for patients with higher GC scores (≥ 0.4), cumulative incidence of metastasis at 5 years was 6% for patients treated with adjuvant RT compared with 23% for patients treated with salvage RT (P < .01). CONCLUSION In patients treated with post-RP RT, GC is prognostic for the development of clinical metastasis beyond routine clinical and pathologic features. Although preliminary, patients with low GC scores are best treated with salvage RT, whereas those with high GC scores benefit from adjuvant therapy. These findings provide the first rational selection of timing for post-RP RT.

Genomic Prostate Cancer Classifier Predicts Biochemical Failure and Metastases in Patients After Postoperative Radiation Therapy

Purpose To test the hypothesis that a genomic classifier (GC) would predict biochemical failure (BF) and distant metastasis (DM) in men receiving radiation therapy (RT) after radical prostatectomy (RP). Methods and Materials Among patients who underwent post-RP RT, 139 were identified for pT3 or positive margin, who did not receive neoadjuvant hormones and had paraffin-embedded specimens. Ribonucleic acid was extracted from the highest Gleason grade focus and applied to a high-density-oligonucleotide microarray. Receiver operating characteristic, calibration, cumulative incidence, and Cox regression analyses were performed to assess GC performance for predicting BF and DM after post-RP RT in comparison with clinical nomograms. Results The area under the receiver operating characteristic curve of the Stephenson model was 0.70 for both BF and DM, with addition of GC significantly improving area under the receiver operating characteristic curve to 0.78 and 0.80, respectively. Stratified by GC risk groups, 8-year cumulative incidence was 21%, 48%, and 81% for BF (P<.0001) and for DM was 0, 12%, and 17% (P=.032) for low, intermediate, and high GC, respectively. In multivariable analysis, patients with high GC had a hazard ratio of 8.1 and 14.3 for BF and DM. In patients with intermediate or high GC, those irradiated with undetectable prostate-specific antigen (PSA ≤0.2 ng/mL) had median BF survival of >8 years, compared with <4 years for patients with detectable PSA (>0.2 ng/mL) before initiation of RT. At 8 years, the DM cumulative incidence for patients with high GC and RTwith undetectable PSA was 3%, compared with 23% with detectable PSA (P=.03). No outcome differences were observed for low GC between the treatment groups. Conclusion The GC predicted BF and metastasis after post-RP irradiation. Patients with lower GC risk may benefit from delayed RT, as opposed to those with higher GC; however, this needs prospective validation. Genomic-based models may be useful for improved decision-making for treatment of high-risk prostate cancer.

Contemporary Update of a Multi-Institutional Predictive Nomogram for Salvage Radiotherapy After Radical Prostatectomy

PURPOSE We aimed to update a previously published, multi-institutional nomogram of outcomes for salvage radiotherapy (SRT) following radical prostatectomy (RP) for prostate cancer, including patients treated in the contemporary era. METHODS Individual data from node-negative patients with a detectable post-RP prostate-specific antigen (PSA) treated with SRT with or without concurrent androgen-deprivation therapy (ADT) were obtained from 10 academic institutions. Freedom from biochemical failure (FFBF) and distant metastases (DM) rates were estimated, and predictive nomograms were generated. RESULTS Overall, 2,460 patients with a median follow-up of 5 years were included; 599 patients (24%) had a Gleason score (GS) ≤ 6, 1,387 (56%) had a GS of 7, 244 (10%) had a GS of 8, and 230 (9%) had a GS of 9 to 10. There were 1,370 patients (56%) with extraprostatic extension (EPE), 452 (18%) with seminal vesicle invasion (SVI), 1,434 (58%) with positive surgical margins, and 390 (16%) who received ADT (median, 6 months). The median pre-SRT PSA was 0.5 ng/mL (interquartile range, 0.3 to 1.1). The 5-yr FFBF rate was 56% overall, 71% for those with a pre-SRT PSA level of 0.01 to 0.2 ng/mL (n = 441), 63% for those with a PSA of 0.21 to 0.50 ng/mL (n = 822), 54% for those with a PSA of 0.51 to 1.0 ng/mL (n = 533), 43% for those with a PSA of 1.01 to 2.0 ng/mL (n = 341), and 37% for those with a PSA > 2.0 ng/mL (n = 323); P < .001. On multivariable analysis, pre-SRT PSA, GS, EPE, SVI, surgical margins, ADT use, and SRT dose were associated with FFBF. Pre-SRT PSA, GS, SVI, surgical margins, and ADT use were associated with DM, whereas EPE and SRT dose were not. The nomogram concordance indices were 0.68 (FFBF) and 0.74 (DM). CONCLUSION Early SRT at low PSA levels after RP is associated with improved FFBF and DM rates. Contemporary nomograms can estimate individual patient outcomes after SRT in the modern era.

Novel actions of next-generation taxanes benefit advanced stages of prostate cancer.

Purpose: To improve the outcomes of patients with castration-resistant prostate cancer (CRPC), there is an urgent need for more effective therapies and approaches that individualize specific treatments for patients with CRPC. These studies compared the novel taxane cabazitaxel with the previous generation docetaxel, and aimed to determine which tumors are most likely to respond. Experimental design: Cabazitaxel and docetaxel were compared via in vitro modeling to determine the molecular mechanism, biochemical and cell biologic impact, and cell proliferation, which was further assessed ex vivo in human tumor explants. Isogenic pairs of RB knockdown and control cells were interrogated in vitro and in xenograft tumors for cabazitaxel response. Results: The data herein show that (i) cabazitaxel exerts stronger cytostatic and cytotoxic response compared with docetaxel, especially in CRPC; (ii) cabazitaxel induces aberrant mitosis, leading to pyknotic and multinucleated cells; (iii) taxanes do not act through the androgen receptor (AR); (iv) gene-expression profiling reveals distinct molecular actions for cabazitaxel; and (v) tumors that have progressed to castration resistance via loss of RB show enhanced sensitivity to cabazitaxel. Conclusions: Cabazitaxel not only induces improved cytostatic and cytotoxic effects, but also affects distinct molecular pathways, compared with docetaxel, which could underlie its efficacy after docetaxel treatment has failed in patients with CRPC. Finally, RB is identified as the first potential biomarker that could define the therapeutic response to taxanes in metastatic CRPC. This would suggest that loss of RB function induces sensitization to taxanes, which could benefit up to 50% of CRPC cases. Clin Cancer Res; 21(4); 795–807. ©2015 AACR.

Characterization of 1577 primary prostate cancers reveals novel biological and clinicopathologic insights into molecular subtypes

BACKGROUND Prostate cancer (PCa) molecular subtypes have been defined by essentially mutually exclusive events, including ETS gene fusions (most commonly involving ERG) and SPINK1 overexpression. Clinical assessment may aid in disease stratification, complementing available prognostic tests. OBJECTIVE To determine the analytical validity and clinicopatholgic associations of microarray-based molecular subtyping. DESIGN, SETTING, AND PARTICIPANTS We analyzed Affymetrix GeneChip expression profiles for 1577 patients from eight radical prostatectomy cohorts, including 1351 cases assessed using the Decipher prognostic assay (GenomeDx Biosciences, San Diego, CA, USA) performed in a laboratory with Clinical Laboratory Improvements Amendment certification. A microarray-based (m-) random forest ERG classification model was trained and validated. Outlier expression analysis was used to predict other mutually exclusive non-ERG ETS gene rearrangements (ETS(+)) or SPINK1 overexpression (SPINK1(+)). OUTCOME MEASUREMENTS Associations with clinical features and outcomes by multivariate logistic regression analysis and receiver operating curves. RESULTS AND LIMITATIONS The m-ERG classifier showed 95% accuracy in an independent validation subset (155 samples). Across cohorts, 45% of PCas were classified as m-ERG(+), 9% as m-ETS(+), 8% as m-SPINK1(+), and 38% as triple negative (m-ERG(-)/m-ETS(-)/m-SPINK1(-)). Gene expression profiling supports three underlying molecularly defined groups: m-ERG(+), m-ETS(+), and m-SPINK1(+)/triple negative. On multivariate analysis, m-ERG(+) tumors were associated with lower preoperative serum prostate-specific antigen and Gleason scores, but greater extraprostatic extension (p<0.001). m-ETS(+) tumors were associated with seminal vesicle invasion (p=0.01), while m-SPINK1(+)/triple negative tumors had higher Gleason scores and were more frequent in Black/African American patients (p<0.001). Clinical outcomes were not significantly different among subtypes. CONCLUSIONS A clinically available prognostic test (Decipher) can also assess PCa molecular subtypes, obviating the need for additional testing. Clinicopathologic differences were found among subtypes based on global expression patterns. PATIENT SUMMARY Molecular subtyping of prostate cancer can be achieved using extra data generated from a clinical-grade, genome-wide expression-profiling prognostic assay (Decipher). Transcriptomic and clinical analysis support three distinct molecular subtypes: (1) m-ERG(+), (2) m-ETS(+), and (3) m-SPINK1(+)/triple negative (m-ERG(-)/m-ETS(-)/m-SPINK1(-)). Incorporation of subtyping into a clinically available assay may facilitate additional applications beyond routine prognosis.

Novel Biomarker Signature That May Predict Aggressive Disease in African American Men With Prostate Cancer

PURPOSE We studied the ethnicity-specific expression of prostate cancer (PC) -associated biomarkers to evaluate whether genetic/biologic factors affect ethnic disparities in PC pathogenesis and disease progression. PATIENTS AND METHODS A total of 154 African American (AA) and 243 European American (EA) patients from four medical centers were matched according to the Cancer of the Prostate Risk Assessment postsurgical score within each institution. The distribution of mRNA expression levels of 20 validated biomarkers reported to be associated with PC initiation and progression was compared with ethnicity using false discovery rate, adjusted Wilcoxon-Mann-Whitney, and logistic regression models. A conditional logistic regression model was used to evaluate the interaction between ethnicity and biomarkers for predicting clinicopathologic outcomes. RESULTS Of the 20 biomarkers examined, six showed statistically significant differential expression in AA compared with EA men in one or more statistical models. These include ERG (P < .001), AMACR (P < .001), SPINK1 (P = .001), NKX3-1 (P = .03), GOLM1 (P = .03), and androgen receptor (P = .04). Dysregulation of AMACR (P = .036), ERG (P = .036), FOXP1 (P = .041), and GSTP1 (P = .049) as well as loss-of-function mutations for tumor suppressors NKX3-1 (P = .025) and RB1 (P = .037) predicted risk of pathologic T3 disease in an ethnicity-dependent manner. Dysregulation of GOLM1 (P = .037), SRD5A2 (P = .023), and MKi67 (P = .023) predicted clinical outcomes, including 3-year biochemical recurrence and metastasis at 5 years. A greater proportion of AA men than EA men had triple-negative (ERG-negative/ETS-negative/SPINK1-negative) disease (51% v 35%; P = .002). CONCLUSION We have identified a subset of PC biomarkers that predict the risk of clinicopathologic outcomes in an ethnicity-dependent manner. These biomarkers may explain in part the biologic contribution to ethnic disparity in PC outcomes between EA and AA men.

Development and validation of a 24-gene predictor of response to postoperative radiotherapy in prostate cancer: a matched, retrospective analysis

BACKGROUND Postoperative radiotherapy has an important role in the treatment of prostate cancer, but personalised patient selection could improve outcomes and spare unnecessary toxicity. We aimed to develop and validate a gene expression signature to predict which patients would benefit most from postoperative radiotherapy. METHODS Patients were eligible for this matched, retrospective study if they were included in one of five published US studies (cohort, case-cohort, and case-control studies) of patients with prostate adenocarcinoma who had radical prostatectomy (with or without postoperative radiotherapy) and had gene expression analysis of the tumour, with long-term follow-up and complete clinicopathological data. Additional treatment after surgery was at the treating physicians discretion. In each cohort, patients who had postoperative radiotherapy were matched with patients who had not had radiotherapy using Gleason score, prostate-specific antigen concentration, surgical margin status, extracapsular extension, seminal vesicle invasion, lymph node invasion, and androgen deprivation therapy. We constructed a matched training cohort using patients from one study in which we developed a 24-gene Post-Operative Radiation Therapy Outcomes Score (PORTOS). We generated a pooled matched validation cohort using patients from the remaining four studies. The primary endpoint was the development of distant metastasis. FINDINGS In the training cohort (n=196), among patients with a high PORTOS (n=39), those who had radiotherapy had a lower incidence of distant metastasis than did patients who did not have radiotherapy, with a 10-year metastasis rate of 5% (95% CI 0-14) in patients who had radiotherapy (n=20) and 63% (34-80) in patients who did not have radiotherapy (n=19; hazard ratio [HR] 0·12 [95% CI 0·03-0·41], p<0·0001), whereas among patients with a low PORTOS (n=157), those who had postoperative radiotherapy (n=78) had a greater incidence of distant metastasis at 10 years than did their untreated counterparts (n=79; 57% [44-67] vs 31% [20-41]; HR 2·5 [1·6-4·1], p<0·0001), with a significant treatment interaction (pinteraction<0·0001). The finding that PORTOS could predict outcome due to radiotherapy treatment was confirmed in the validation cohort (n=330), which showed that patients who had radiotherapy had a lower incidence of distant metastasis compared with those who did not have radiotherapy, but only in the high PORTOS group (high PORTOS [n=82]: 4% [95% CI 0-10] in the radiotherapy group [n=57] vs 35% [95% CI 7-54] in the no radiotherapy group [n=25] had metastasis at 10 years; HR 0·15 [95% CI 0·04-0·60], p=0·0020; low PORTOS [n=248]: 32% [95% CI 19-43] in the radiotherapy group [n=108] vs 32% [95% CI 22-40] in the no radiotherapy group [n=140]; HR 0·92 [95% CI 0·56-1·51], p=0·76), with a significant interaction (pinteraction=0·016). The conventional prognostic tools Decipher, CAPRA-S, and microarray version of the cell cycle progression signature did not predict response to radiotherapy (pinteraction>0·05 for all). INTERPRETATION Patients with a high PORTOS who had postoperative radiotherapy were less likely to have metastasis at 10 years than those who did not have radiotherapy, suggesting that treatment with postoperative radiotherapy should be considered in this subgroup. PORTOS should be investigated further in additional independent cohorts. FUNDING None.