Repeat Next-Generation Sequencing Testing on Progression in Men With Metastatic Prostate Cancer Can Identify New Actionable Alterations.

PURPOSE: There are limited data available on the real-world patterns of molecular testing in men with advanced prostate cancer. We thus sought to evaluate next-generation sequencing (NGS) testing in the United States, focused on single versus serial NGS testing, the different disease states of testing (hormone-sensitive v castration-resistant, metastatic vs nonmetastatic), tissue versus plasma circulating tumor DNA (ctDNA) assays, and how often actionable data were found on each NGS test. METHODS: The Prostate Cancer Precision Medicine Multi-Institutional Collaborative Effort clinical-genomic database was used for this retrospective analysis, including 1,597 patients across 15 institutions. Actionable NGS data were defined as including somatic alterations in homologous recombination repair genes, mismatch repair deficiency, microsatellite instability (MSI-high), or a high tumor mutational burden ≥10 mut/MB. RESULTS: Serial NGS testing (two or more NGS tests with specimens collected more than 60 days apart) was performed in 9% (n = 144) of patients with a median of 182 days in between test results. For the second NGS test and beyond, 82.1% (225 of 274) of tests were from ctDNA assays and 76.1% (217 of 285) were collected in the metastatic castration-resistant setting. New actionable data were found on 11.1% (16 of 144) of second NGS tests, with 3.5% (5 of 144) of tests detecting a new BRCA2 alteration or MSI-high. A targeted therapy (poly (ADP-ribose) polymerase inhibitor or immunotherapy) was given after an actionable result on the second NGS test in 31.3% (5 of 16) of patients. CONCLUSION: Repeat somatic NGS testing in men with prostate cancer is infrequently performed in practice and can identify new actionable alterations not present with initial testing, suggesting the utility of repeat molecular profiling with tissue or blood of men with metastatic castration-resistant prostate cancer to guide therapy choices.

Biomarker-Directed Therapy in Black and White Men With Metastatic Castration-Resistant Prostate Cancer.

Importance: Black men have higher incidence and mortality from prostate cancer. Whether precision oncology disparities affect Black men with metastatic castration-resistant prostate cancer (mCRPC) is unknown. Objective: To compare precision medicine data and outcomes between Black and White men with mCRPC. Design, Setting, and Participants: This retrospective cohort study used data collected by the Prostate Cancer Precision Medicine Multi-Institutional Collaborative Effort (PROMISE) consortium, a multi-institutional registry with linked clinicogenomic data, from April 2020 to December 2021. Participants included Black and White patients with mCRPC with molecular data. Data were analyzed from December 2021 to May 2023. Exposures: Database-reported race and ethnicity. Main Outcomes and Measures: The primary outcome was the frequency of actionable molecular data, defined as the presence of mismatch repair deficiency (MMRD) or high microsatellite instability (MSI-H), homologous recombination repair deficiency, or tumor mutational burden of 10 mutations per megabase or greater. Secondary outcomes included the frequency of other alterations, the type and timing of genomic testing performed, and use of targeted therapy. Efficacy outcomes were prostate-specific antigen response rate, site-reported radiographic response, and overall survival. Results: A total of 962 eligible patients with mCRPC were identified, including 204 Black patients (21.2%; median [IQR] age at diagnosis, 61 [55-67] years; 131 patients [64.2%] with Gleason scores 8-10; 92 patients [45.1%] with de novo metastatic disease) and 758 White patients (78.8%; median [IQR] age, 63 [57-69] years; 445 patients [58.7%] with Gleason scores 8-10; 310 patients [40.9%] with de novo metastatic disease). Median (IQR) follow-up from mCRPC was 26.6 (14.2-44.7) months. Blood-based molecular testing was more common in Black men (111 men [48.7%]) than White men (317 men [36.4%]; P < .001). Rates of actionable alterations were similar between groups (65 Black men [32.8%]; 215 White men [29.1%]; P = .35), but MMRD or MSI-H was more common in Black men (18 men [9.1]) than White men (36 men [4.9%]; P = .04). PTEN alterations were less frequent in Black men than White men (31 men [15.7%] vs 194 men [26.3%]; P = .003), as were TMPRSS alterations (14 men [7.1%] vs 155 men [21.0%]; P < .001). No other differences were seen in the 15 most frequently altered genes, including TP53, AR, CDK12, RB1, and PIK3CA. Matched targeted therapy was given less frequently in Black men than White men (22 men [33.5%] vs 115 men [53.5%]; P = .008). There were no differences in response to targeted therapy or survival between the two cohorts. Conclusions and Relevance: This cohort study of men with mCRPC found higher frequency of MMRD or MSI-H and lower frequency of PTEN and TMPRSS alterations in Black men compared with White men. Although Black men received targeted therapy less frequently than White men, no differences were observed in clinical outcomes.

Race-Associated Genomic Correlates of Therapeutic Response in African American Patients With Non-Small-Cell Lung Cancer.

PURPOSE: African American individuals are disproportionately affected by lung cancer in terms of incidence and mortality. In oncogene-driven non-small-cell lung cancer (NSCLC), emerging evidence indicates that underlying molecular heterogeneity, which can be affected by ancestry, contributes to variable drug sensitivity and therapeutic responses. The purpose of this study was to evaluate race-associated differences in reported treatment decisions, therapeutic outcomes, and molecular features in KRAS- and EGFR-mutant NSCLC. MATERIALS AND METHODS: This is a retrospective study using real-world clinical-genomic data from health systems in the United States to evaluate race-associated outcomes in advanced-stage KRAS- or EGFR-driven NSCLC. Our overall objectives were to evaluate race-associated therapeutic outcomes and to describe molecular features in non-Hispanic Black (NHB) and non-Hispanic White (NHW) patients with NSCLC. RESULTS: A total of 723 NSCLC patients with KRAS and 315 patients with EGFR oncogenic mutations were evaluated. In KRAS-mutant patients, variable outcomes were observed in NHB and NHW patients on the basis of receiving chemotherapy alone or in combination with immune checkpoint inhibitors. NHB patients received treatment at significantly lower rates compared with NHW patients. In the EGFR-mutant cohort, NHB and NHW patients received EGFR-targeted agents at similar rates, and overall survival was not significantly different. Race-associated differences in molecular features included a higher frequency of TP53 comutation in KRAS-mutant NHB patients and higher prevalence of EGFR G719S subtype in NHB patients. CONCLUSION: In a real-world cohort of patients with NSCLC, we identified race-associated differences in therapeutic outcomes and described molecular characteristics in NHB and NHW patients with NSCLC. To proactively identify patients most likely to respond to systemic therapies, a more comprehensive approach is needed to help guide therapy selection in individualized patient populations.

Molecular features and race-associated outcomes of SPOP-mutant metastatic castration-resistant prostate cancer.

BACKGROUND: Inactivating alterations in SPOP frequently occur in prostate cancer and promote increased dependency on androgen receptor (AR)-mediated oncogenic signaling. The presence of SPOP mutation (SPOP-mutant [SPOP-mut]) may therefore impact therapeutic outcomes with AR-directed therapies and docetaxel in metastatic castration-resistant (mCRPC). METHODS: This was a retrospective study of mCRPC patients treated at an urban academic hospital (n = 103). Patients underwent tumor DNA sequencing to determine SPOP mutational status (SPOP-mut). Outcomes measured were overall survival (OS) from diagnosis and treatment with second-generation AR signaling inhibitor (ARSI) or docetaxel and time to PSA progression (prostate-specific antigen-progression-free survival [PSA-PFS]) compared by SPOP status using Kaplan-Meier curves and log-rank test. The univariable and multivariable Cox proportional hazard model evaluated the association of SPOP mutation and outcomes adjusted for clinicopathologic features. RESULTS: SPOP-mut was associated with longer PSA-PFS in mCRPC (median 1.79 vs. 0.84 years; p = 0.06) and multivariate analysis (hazard ratio [HR] = 0.37; 95% confidence interval [CI]: 0.17-0.84; p = 0.02). SPOP-mut demonstrated a higher median PSA decline compared to SPOP wild-type (median decline 100% vs. 92%, p = 0.02). SPOP-mut was not associated with OS from the start of ARSI or docetaxel (median OS not reached vs. 2.0 years) or PSA-PFS on docetaxel (median PSA-PFS 0.4 vs. 0.5 years) in mCRPC. The majority of SPOP mutations were identified in African American (AA) patients (69.2%) compared to Caucasian patients (30.8%). Race-associated multivariate analysis revealed no significant differences in OS from the start of ARSI or the start of docetaxel and no differences in ARSI or docetaxel PSA-PFS between AA and Caucasian patients. Molecular profiling demonstrated that AA patients had a higher frequency of SPOP mutations and greater heterogeneity of SPOP variants within the coding sequence. Analysis of concurrent genomic alterations revealed that SPOP mutations co-occur with APC mutations (p = 0.001) and alterations in the Wnt pathway (p = 0.017). CONCLUSIONS: Inactivating mutations in SPOP are associated with better response to ARSI treatment in mCRPC overall. Additional analysis with a larger cohort is needed to evaluate the association of SPOP status and outcomes with docetaxel. Race-associated clinical outcomes and molecular features were observed, suggesting the benefit of biomarker-directed therapy selection for individualized patient subsets in guiding treatment decisions for mCRPC patients.

Androgen receptor negatively regulates mitotic checkpoint signaling to induce docetaxel resistance in castration-resistant prostate cancer.

BACKGROUND: Despite multiple treatment advances for castration-resistant prostate cancer (CRPC), there are currently no curative therapies and patients ultimately to succumb to the disease. Docetaxel (DTX) is the standard first-line chemotherapy for patients with metastatic CRPC; however, drug resistance is inevitable and often develops rapidly, leading to disease progression in nearly all patients. In contrast, when DTX is deployed with androgen deprivation therapy in castration-sensitive disease, more durable responses and improved outcomes are observed, suggesting that aberrant androgen receptor (AR) signaling accelerates DTX resistance in CRPC. In this study, we demonstrate that AR dysregulates the mitotic checkpoint, a critical pathway involved in the anticancer action of DTX. METHODS: Androgen-dependent and independent cell lines were used to evaluate the role of AR in DTX resistance. Impact of drug treatment on cell viability, survival, and cell-cycle distribution were determined by plate-based viability assay, clonogenic assay, and cell-cycle analysis by flow cytometry, respectively. Mitotic checkpoint kinase signal transduction and apoptosis activation was evaluated by Western blotting. Pathway gene expression analysis was evaluated by RT-PCR. A Bliss independence model was used to calculate synergy scores for drug combination studies. RESULTS: Activation of AR in hormone-sensitive cells induces a rescue phenotype by increasing cell viability and survival and attenuating G2/M arrest in response to DTX. Analysis of mitotic checkpoint signaling shows that AR negatively regulates spindle checkpoint signaling, resulting in premature mitotic progression and evasion of apoptosis. This phenotype is characteristic of mitotic slippage and is also observed in CRPC cell lines where we demonstrate involvement of AR splice variant AR-v7 in dysregulation of checkpoint signaling. Our findings suggest that DTX resistance is mediated through mechanisms that drive premature mitotic exit. Using pharmacologic inhibitors of anaphase-promoting complex/cyclosome and polo-like kinase 1, we show that blocking mitotic exit induces mitotic arrest, apoptosis, and synergistically inhibits cell survival in combination with DTX. CONCLUSION: Our results suggest that targeting the mechanisms of dysregulated mitotic checkpoint signaling in AR-reactivated tumors has significant clinical potential to extend treatment benefit with DTX and improve outcomes in patients with lethal prostate cancer.

PROMISE: a real-world clinical-genomic database to address knowledge gaps in prostate cancer.

PURPOSE: Prostate cancer is a heterogeneous disease with variable clinical outcomes. Despite numerous recent approvals of novel therapies, castration-resistant prostate cancer remains lethal. A "real-world" clinical-genomic database is urgently needed to enhance our characterization of advanced prostate cancer and further enable precision oncology. METHODS: The Prostate Cancer Precision Medicine Multi-Institutional Collaborative Effort (PROMISE) is a consortium whose aims are to establish a repository of de-identified clinical and genomic patient data that are linked to patient outcomes. The consortium structure includes a (1) bio-informatics committee to standardize genomic data and provide quality control, (2) biostatistics committee to independently perform statistical analyses, (3) executive committee to review and select proposals of relevant questions for the consortium to address, (4) diversity/inclusion committee to address important clinical questions pertaining to racial disparities, and (5) patient advocacy committee to understand patient perspectives to improve patients' quality of care. RESULTS: The PROMISE consortium was formed by 16 academic institutions in early 2020 and a secure RedCap database was created. The first patient record was entered into the database in April 2020 and over 1000 records have been entered as of early 2021. Data entry is proceeding as planned with the goal to have over 2500 patient records by the end of 2021. CONCLUSIONS: The PROMISE consortium provides a powerful clinical-genomic platform to interrogate and address data gaps that have arisen with increased genomic testing in the clinical management of prostate cancer. The dataset incorporates data from patient populations that are often underrepresented in clinical trials, generates new hypotheses to direct further research, and addresses important clinical questions that are otherwise difficult to investigate in prospective studies.

The Potential and Limitations of Precision Oncology: Lessons Learned from Whole-Exome Sequencing in an Exceptional Response to Everolimus in Advanced Renal Cell Carcinoma.

Through elucidating the genetic mechanisms of drug sensitivity, precision medicine aims to improve patient selection and response to therapy. Exceptional responders are patients that exhibit exquisite and durable responses to targeted therapy, providing a rare opportunity to identify the molecular basis of drug sensitivity. We identified an exceptional responder to everolimus, an oral inhibitor of the mammalian target of rapamycin (mTOR) pathway, in a patient with advanced renal cell carcinoma. Through whole-exome sequencing on pretreatment and metastatic tumor DNA, we identified alterations in several mTOR pathway genes, with several mutations implicated in mTOR activation. Importantly, these alterations are currently not included in commercially available next-generation sequencing panels, suggesting that precision medicine is still limited in its ability to predict responses to mTOR-targeted therapies. Further research to discover and validate predictive biomarkers of response to everolimus and other targeted therapies is urgently needed. Given the rarity of patients with exceptional responses to targeted agents, cooperative efforts to understand the molecular basis for these phenotypes are essential.

Targeting prosurvival BCL2 signaling through Akt blockade sensitizes castration-resistant prostate cancer cells to enzalutamide.

BACKGROUND: Prostate cancer that recurs after initial treatment inevitably progresses to castration-resistant prostate cancer (CRPC), the lethal stage of the disease. Despite improvements in outcomes from next generation androgen receptor (AR)-axis inhibitors, CRPC remains incurable. Therapeutic strategies to target AR antagonist resistance are urgently needed to improve outcomes for men with this lethal form of prostate cancer. METHODS: Apoptosis and BCL2 family signaling were characterized in cell line models of CRPC. Quantitative real-time polymerase chain reaction and Western blot analysis were used to determine BCL2 expression levels. Drug sensitivity was determined by proliferation, survival and apoptosis analysis. Protein-protein interactions were evaluated by coimmunoprecipitation followed by Western blot detection. RESULTS: In the present study, we identify antiapoptotic BCL2 protein signaling as a mechanism of resistance to AR antagonist enzalutamide. In CRPC cell line models, we found that BCL-xL and MCL-1 proteins block apoptosis through binding and sequestering proapoptotic proteins BIM and BAX, resulting in cell survival in response to enzalutamide. Treatment with BH3-mimetics targeting BCL-xL or MCL-1 disrupts these interactions and activates apoptosis, sensitizing CRPC cells to enzalutamide. Importantly, we demonstrate that PI3K/Akt signaling is activated in response to enzalutamide and mediates apoptosis evasion through inactivation of BAD, a BH3-only protein that activates proapoptotic signlaing through inhbition of BCL-xL. Inhibition of Akt activates BAD, resulting in increased apoptosis and sensitivity to enzalutamide, demonstrating an alternative therapeutic strategy to target drug resistance. CONCLUSIONS: These results demonstrate that CRPC cells employ multiple mechanisms to mediate apoptosis evasion through BCL2 signaling, suggesting this pathway is critical for survival. This study provides a strong preclinical rationale for developing therapeutic strategies to target antiapoptotic BCL2 signaling in combination with AR antagonists to improve treatment options for patients with advanced prostate cancer.

ALK is a critical regulator of the MYC-signaling axis in ALK positive lung cancer.

A subset of lung cancers is dependent on the anaplastic lymphoma kinase (ALK) oncogene for survival, a mechanism that is exploited by the use of the ALK inhibitor crizotinib. Despite exceptional initial tumor responses to ALK inhibition by crizotinib, durable clinical response is limited and the emergence of drug resistance occurs. Furthermore, intrinsic resistance is frequently observed, where patients fail to respond initially to ALK-inhibitor therapy. These events demonstrate the underlying complexity of a molecularly-defined oncogene-driven cancer and highlights the need to identify compensating survival pathways. Using a loss-of-function whole genome short-hairpin (shRNA) screen, we identified MYCBP as a determinant of response to crizotinib, implicating the MYC signaling axis in resistance to crizotinib-treated ALK+ NSCLC. Further analysis reveals that ALK regulates transcriptional expression of MYC and activates c-MYC transactivation of c-MYC target genes. Inhibition of MYC by RNAi or small molecules sensitizes ALK+ cells to crizotinib. Taken together, our findings demonstrate a dual oncogene mechanism, where ALK positively regulates the MYC signaling axis, providing an additional oncogene target whose inhibition may prevent or overcome resistance.

IAP Antagonists Enhance Apoptotic Response to Enzalutamide in Castration-Resistant Prostate Cancer Cells via Autocrine TNF-α Signaling.

BACKGROUND: Castration-resistant prostate cancer (CRPC) remains incurable and identifying effective treatments continues to present a clinical challenge. Although treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, prolongs survival in prostate cancer patients, responses can be limited by intrinsic resistance or acquired resistance. A potential mechanism of resistance to androgen axis inhibition is evasion of apoptosis. Inhibitor of apoptosis proteins (IAPs) are found to be overexpressed in prostate cancer and function to block apoptosis and promote survival signaling. Novel, small-molecule IAP antagonists, such as AEG40995, are emerging as a strategy to induce apoptosis and increase therapeutic response in cancer. METHODS: Human prostate cancer cell lines LNCaP and C4-2 were treated with enzalutamide with or without addition of IAP antagonist AEG40995 and proliferation and survival were determined by MTS and clonogenic assay. Western blot was used to evaluate IAP protein expression changes and PARP-1 cleavage was assessed as indication of apoptosis. Flow cytometry was performed to analyze apoptosis in treated cells. Caspase activity was determined by luminescence assay. Quantitative real-time PCR and immunometric ELISA was used to assess TNF-α (transcript and protein levels, respectively) in response to treatment. RESULTS: In this study, we demonstrate that IAP antagonist AEG40995 exhibits minimal effects on prostate cancer cell proliferation or survival, but rapidly degrades cIAP1 protein. Combination treatment with enzalutamide demonstrates that AEG40995 increases apoptosis and reduces proliferation and clonogenic survival in cell line models of prostate cancer. Mechanistically, we demonstrate that apoptosis in response to enzalutamide and IAP antagonist requires activation of caspase-8, suggesting extrinsic/death receptor apoptosis signaling. Assessment of TNF-α in response to combination treatment with enzalutamide and AEG40995 reveals increased mRNA expression and autocrine protein secretion. Blocking TNF-α signaling abrogates the apoptotic response demonstrating that TNF-α plays a critical role in executing cell death in response to this drug combination. CONCLUSIONS: These findings suggest that IAP antagonists can increase sensitivity and amplify the caspase-mediated apoptotic response to enzalutamide through TNF-α signaling mechanisms. Combination with an IAP antagonist increases enzalutamide sensitivity, lowers the apoptotic threshold and may combat drug resistance in patients with prostate cancer. Prostate 77:866-877, 2017. © 2017 Wiley Periodicals, Inc.

Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer.

PURPOSE: Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small-molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in patients with ALK(+) non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: We analyzed tissue obtained from 14 patients with ALK(+) NSCLC showing evidence of radiologic progression while on crizotinib to define mechanisms of intrinsic and acquired resistance to crizotinib. RESULTS: Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient showed outgrowth of epidermal growth factor receptor (EGFR) mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient showed the emergence of an ALK gene fusion-negative tumor compared with the baseline sample but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism. CONCLUSIONS: Crizotinib resistance in ALK(+) NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers.

Folate deficiency regulates expression of DNA polymerase ß in response to oxidative stress.

Folate deficiency has been shown to influence carcinogenesis by creating an imbalance in the base excision repair (BER) pathway, affecting BER homeostasis. The inability to mount a BER response to oxidative stress in a folate-deficient environment results in the accumulation of DNA repair intermediates, i.e., DNA strand breaks. Our data indicate that upregulation of ß-pol expression in response to oxidative stress is inhibited by folate deficiency at the level of gene expression. Alteration in the expression of ß-pol in a folate-deficient environment is not due to epigenetic changes in the core promoter of the ß-pol gene, i.e., the CpG islands within the ß-pol promoter remain unmethylated in the presence or absence of folate. However, the promoter analysis studies show a differential binding of regulatory factors to the -36 to -7 region (the folic acid-response region, FARR) within the core promoter of ß-pol. Moreover, we observe a tight correlation between the level of binding of regulatory factors with the FARR and inhibition of ß-pol expression. Based on these findings, we propose that folate deficiency results in an upregulation/stability of negative regulatory factors interacting with FARR, repressing the upregulation of the ß-pol gene in response to oxidative stress.

Folate deficiency provides protection against colon carcinogenesis in DNA polymerase beta haploinsufficient mice.

Aging and DNA polymerase beta deficiency (beta-pol(+/-)) interact to accelerate the development of malignant lymphomas and adenocarcinoma and increase tumor bearing load in mice. Folate deficiency (FD) has been shown to induce DNA damage repaired via the base excision repair (BER) pathway. We anticipated that FD and BER deficiency would interact to accelerate aberrant crypt foci (ACF) formation and tumor development in beta-pol haploinsufficient animals. FD resulted in a significant increase in ACF formation in wild type (WT) animals exposed to 1,2-dimethylhydrazine, a known colon and liver carcinogen; however, FD reduced development of ACF in beta-pol haploinsufficient mice. Prolonged feeding of the FD diet resulted in advanced ACF formation and liver tumors in wild type mice. However, FD attenuated onset and progression of ACF and prevented liver tumorigenesis in beta-pol haploinsufficient mice, i.e. FD provided protection against tumorigenesis in a BER-deficient environment in all tissues where 1,2-dimethylhydrazine exerts its damage. Here we show a distinct down-regulation in DNA repair pathways, e.g. BER, nucleotide excision repair, and mismatch repair, and decline in cell proliferation, as well as an up-regulation in poly(ADP-ribose) polymerase, proapoptotic genes, and apoptosis in colons of FD beta-pol haploinsufficient mice.