AR Mutations Research Articles

Genetic profiling of hormone-sensitive and castration-resistant prostate cancers and identification of genetic mutations prone to castration-resistant prostate cancer.

Genetic profiling of patients with prostate cancer could potentially identify mutations prone to castration-resistant prostate cancer (CRPC). Here, we aimed to identify the differences in genetic profiles of patients with hormone-sensitive prostate cancer (HSPC) and CRPC and stratify HSPC patients to identify mutations associated with CRPC progression. A total of 103 samples were collected, including 62 DNA samples from the tumor tissues of 59 HSPC patients and 41 cell-free DNA (cfDNA) samples from prostate cancer patients at different cancer stages. Targeted sequence was conducted on both the tissue DNA and cfDNA. The associations between mutations and clinical outcomes (CRPC-free time) were analyzed using χ2 test, logistic regression analysis, Kaplan-Meier analysis, and Cox regression analysis. By comparing to that of cfDNA sequencing, the results from DNA sequencing of 1-needle (80%) and mixed 12-needle (77.8%) biopsies are highly comparable. FOXA1 (30.5%), CDK12 (23.7%), and TP53 (22.0%) were the top 3 most frequently mutated genes in HSPC patients; 50.8% (30/59) and 44.1% (26/59) HSPC patients had mutations in DDR and HRR pathway, respectively. Mutations in AR and APC as well as the members involved in the regulation of stem cell pluripotency and EMT pathway were often observed in CRPC samples. We established a panel of four genetic mutations (MSH2, CDK12, TP53, and RB1) to predict the risk of CRPC early progression with concordance index = 0.609 and the area under curve of the ROC curve as 0.838. In this study, we demonstrated that the cfDNA can be used in genetic profiling in prostate cancer and our newly established panel is capable of predicting which mHSPC patient has a high risk of early CRPC progression.

OR05-1 Testicular Magnetic Resonance Imaging (MRI) Findings and Predictive Factors of Gonadal Neoplasia in Complete Androgen Insensitivity Syndrome

Abstract Introduction Complete Androgen Insensitivity Syndrome (CAIS) is characterized by a complete external genitalia appearance and testicular development in 46,XY individuals carrying inactivating mutations in the AR gene. Prophylactic gonadectomy versus saving gonads has been debatable in CAIS management, mainly due to the absence of an accurate biomarker for testicular germ cell tumors (GCT). Objective to cross the preoperative pelvic MRI data with the histological gonadal findings from CAIS patients who underwent prophylactic gonadectomy to identify predictive factors of GCT development. Methods We evaluated 30 gonads from 15 CAIS patients with proven AR mutation. All performed pelvic MRI before gonadectomy through the same MRI machine and were included in the MRI data analysis. Three blinded radiologists specialized in urology performed image analysis. The immunohistochemical markers PLAP, c-KIT, OCT3/4, SALL4, ER, alpha-inhibin, CD99, and calretinin were performed. Results Nine (60%) performed gonadectomy after puberty (>16 years old). We identified one case of classical seminoma and one case of Sertoli cell neoplasia (at 18 and 19 years of age, respectively). Overall, the prevalence of neoplasia was 13.3%. No case of premalignant intratubular germ cell neoplasia was detected.Histological data revealed a high prevalence of benign lesions (n=12; 80%). Leydig Cell Hyperplasia (67%), stromal fibrosis (53%), Sertoli Cell nodules (47%), hamartomatoums nodules (40%) and paratesticular cysts (67%) which are more frequent after puberty (p<.01). MRI detected heterogeneity in all cases harboring hamartomatoums nodules, Sertoli cell nodules, and testicular neoplasia. Benign lesions were bilateral in all cases. Regarding the MRI data, the presence of testicular cystic mass (by tumoral necrosis) on MRI and tumor size (higher among malignancies) correlated with malignancy (p=.008 and p=.03, respectively), whereas gonadal location (inguinal/abdominal), presence of solid nodules, T2WI and postcontrast features, nodule diffusion restriction, and presence of perigonadal cysts did not. Chronological age >16 years was associated with malignancy with a 4.3 OR (1.6–11.69). Conclusion Pelvic MRI is sensitive to detect gonadal nodules in CAIS patients, regardless of the gonad position, but most nodules are benign. Testicular malignancy should be suspicious in the presence of testicular cystic mass and large testis size in CAIS patients >16 years old. Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.

DICER1-associated central nervous system sarcoma with neural lineage differentiation: a case report

BackgroundDICER1-associated central nervous system sarcoma (DCS) without evidence of other cancer-related syndromes is rare. Though the morphology of DCS was highly variable, the immunophenotype was predominant myogenic phenotype. Other lineage markers were consistently negative.Case presentationWe report a case of DCS with neurogenic differentiation proved by immunohistochemical staining and whole-exome sequencing (WES). An 8-year-old female patient presented with 8-day history of headache, nausea and vomiting. Magnetic resonance imaging (MRI) revealed a heterogeneous mass in the left parietal lobe. The patient underwent the craniotomy via left parietal approach to resect the tumor completely. Histologically, the tumor predominately showed fibrosarcoma-like spindle cells with obvious cytoplasmic eosinophilic globules. Immunohistochemically, the tumor stained positively for DICER1, Desmin, and several neurogenic markers. DICER1 somatic hotspot mutation was confirmed by WES, as well as TP53 and RAF1 mutations which were commonly found in DCS, and other sarcoma-associated genes including AR, AXL and ETV5 mutations. Subsequently, the result of Gene Ontology (GO) analysis showed that the mutated genes in this case were involved in neuron development. All of these findings indicated the diagnosis of DCS with neurogenic differentiation. Postoperatively, the patient received high-dose radiotherapy (60 Gy) and chemotherapy. There was no MRI evidence of tumor recurrence at the 21-month postoperative follow-up.ConclusionsThis unusual DCS case with neuronal differentiation is an important addition to the immuno-phenotypic spectrum of DCS. Although the prognosis for DCS is poor, gross tumor resection with high dose radiotherapy and chemotherapy may assist in prolonging survival.

Identification of UBA1 as the causative gene of an X-linked non-Kennedy spinal-bulbar muscular atrophy.

Spinal-bulbar muscular atrophy (SBMA) (Kennedy's disease) is a motor neuron disease. Kennedy's disease is nearly exclusively caused by mutations in the androgen receptor encoding gene (AR). The results of studies aimed at identification of the genetic cause of a disease that best approximates SBMA in a pedigree (four patients) without mutations in AR are reported. Clinical investigations included thorough neurological and non-neurological examinations and testing. Genetic analysis was performed by exome sequencing using standard protocols. UBA1 mutations were modeled on the crystal structure of UBA1. The clinical features of the patients are described in detail. A missense mutation in UBA1 (c.T1499C; p.Ile500Thr) was identified as the probable cause of the non-Kennedy SBMA in the pedigree. Like AR, UBA1 is positioned on chromosome X. UBA1 is a highly conserved gene. It encodes ubiquitin-like modifier activating enzyme 1 (UBA1) which is the major E1 enzyme of the ubiquitin-proteasome system. Interestingly, UBA1 mutations can also cause infantile-onset X-linked spinal muscular atrophy (XL-SMA). The mutation identified here and the XL-SMA causative mutations were shown to affect amino acids positioned in the vicinity of UBA1's ATP binding site and to cause structural changes. UBA1 was identified as a novel SBMA causative gene. The gene affects protein homeostasis which is one of most important components of the pathology of neurodegeneration. The contribution of this same gene to the etiology of XL-SMA is discussed.

196P Molecular profiling to identify recurrent mutations comparing pre and post-neoadjuvant chemotherapy tumour specimens of TNBC patients with residual disease

The change in mutational landscape of TNBC patients with residual disease after neoadjuvant chemotherapy(NAC) might provide valuable insights into its biology and provide targets for therapy. TNBC patients with residual disease after anthracycline taxane NAC +/- platinum were included. Archival paired (pre and post NAC) samples were processed for NGS with a custom-designed 72-gene panel. The sequences were aligned to human reference genome (GRCh37/hg19) using BWA program. Somatic mutations identified using UMI corrected .clc pipeline and LoFreq (v2) variant caller. Mutations annotated using in-house annotation pipeline (VariMAT). Gene annotation of the variants was performed using VeP program against the Ensembl release 90 human gene Model. 25 patients treated from 2018-2019 were included. 72% patients had stage III disease. Among all 50 tumour samples (pre and post NAC), a median of 12 mutations per sample was observed. 73.5% were missense mutations. Post Platinum NAC an average of 12.8±7 gene mutations were seen and 11.3±5 gene mutations without platinum(p 0.94). Following platinum-NAC, the common genes mutated were TP53(81%), AR(62%), PTEN(62%), PMS2(43%) and ERBB2(33%). In post-NAC samples, private mutations were present only in the platinum group. The major differences between no platinum vs. platinum group was seen with AKT1 (50 vs. 14.3%; p 0.17), MSH6(50 vs. 14.3%; p 0.17), ERBB2(75 vs. 33.3%; p 0.16), HRAS(25 vs. 4.8%; p 0.3) and STK11 (0 vs. 33.3%; p 0.24). Five patients had recurrence of breast cancer. Post-NAC they had on average 14±7 mutated genes vs. 12.2±6 in those recurrence-free (p 0.49).Table: 196PProportion of patients with mutationsPre-NACPost-NACTP53(80%)TP53(84%)PMS2(64%)AR(60%)PTEN(64%)PTEN(60%)ERBB2(48%)PMS2(44%)NOTCH1(44%)ERBB2(40%)Recurrence-freeRecurrent diseaseTP53(85%)TP53(80%)PTEN(65%)AR(60%)AR(60%)ERBB2(60%)PMS2(45%)PMS2(45%)SMARCA4(40%)AKT1(40%) Open table in a new tab The somatic mutational profile was distinct comparing pre vs. post-NAC tumour samples, platinum vs. non-platinum NAC and recurrence vs. no recurrence. AR mutations were present in a higher proportion post-NAC. These findings need further elucidation in larger prospective studies.

Identification of the gossypol derivatives as androgen receptor inhibitor

Prostate cancer (PCa) is the most frequently diagnosed male malignant tumor and remains the second leading cause of male cancer mortality in western countries. The development of novel antiandrogens to circumvent the drug resistance in anti-PCa treatment is highly demanded. Herein, we identified that gossypol (GOS) specificly inhibited the AR signaling. Further optimization of GOS derivatives led to the discovery of compound XY-32. XY-32 efficiently inhibits AR signaling with the IC50 of 1.23 μM. XY-32 downregulates both the full-length AR and the AR variable splice AR-V7 via suppressing the mRNA expression. It inhibits the proliferation of CRPC cells such as the LNCaP cells, the PC-3 cells, and enzalutamide resistance 22Rv1 cells. The work demonstrates the GOS derivatives represent a novel series of anti-androgen to conquer the acquired AR mutations or AR splice variants induced drug resistance of mCRPC.

Abstract 3109: Establishment and characterization of a panel of castrate-resistant prostate cancer XPDX models with differential enzalutamide response

Abstract Background: Preclinical models of prostate cancer are challenging to develop and maintain, especially those that grow in a castrated setting while maintaining receptor and antigen expression. To this end, we have established and characterized a panel of XenoSTART patient-derived (XPDX) prostate models using both intact and castrated athymic nude mice. These models, designated ST1273, ST2347, ST4017, and ST4420, were characterized for receptor expression, genomic alterations, and in vivo drug sensitivity to relevant therapies. Methods: XPDX models representing prostate cancer were established from primary (ST1273) or metastatic (ST2347, ST4017, ST4420) biopsy samples implanted into intact athymic nude mice supplemented with exogenous testosterone. Resulting models were passaged and further developed in both intact and castrated athymic nudes until growth stabilization. Resulting models were characterized using genomic analysis, including WES and RNAseq, receptor expression, and in vivo drug sensitivity studies. Models found sensitive to enzalutamide were conditioned to resistance in vivo by chronic drug administration and resulting models (designated STxxxx/EZR) were characterized and compared with parent lines. For in vivo studies, activity of relevant treatments were benchmarked including enzalutamide administered once daily by oral gavage at 50 mg/kg and docetaxel administered by intravenous injection once weekly at 10 mg/kg. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Results: Each model developed in a castrate or conditioned setting retained similar receptor expression to the parent model including positive AR (2+/3+) and PSMA (2+/3+) staining. Genomic characterization identified a PIK3CA mutation in ST1273 models (PIK3CAE545A), AR mutations in ST2347 (ART878A) and ST4017 (ARH875Y), and a TMPRSS2:ERG fusion in ST4420. In vivo, the ST1273 parent model was found sensitive to enzalutamide (%T/C=11%) but insensitive in castrated mice (%T/C=76%) and the ST1273/EZR model was resistant to enzalutamide in intact (%T/C=56%) or castrated (%T/C=100%) mice. The ST2347 parent model was also found sensitive to enzalutamide (%T/C=19%) but insensitive in castrated mice (%T/C=50%). ST4017 and ST4420 studies in intact and castrated mice are currently underway. All models were sensitive to docetaxel. Conclusion: We have established and characterized a panel of prostate XPDX models using both intact and castrated athymic nude mice and conditioned resistance to enzalutamide by chronic drug administration. These models can be utilized as a valuable tool in better understanding castrate-resistant prostate cancer and in developing novel therapies for enzalutamide-resistant patients. Citation Format: Johnnie Flores, Alyssa Simonson, Dustin Kneifel, Alejandra Diaz, Morgan Harris, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Scott Ulmer, Michael J. Wick. Establishment and characterization of a panel of castrate-resistant prostate cancer XPDX models with differential enzalutamide response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3109.

Variable detection of actionable alterations across racial groups and association with testing patterns.

3116 Background: Molecular landscape studies are critical to biomarker discovery and precision oncology research. However, non-White patients (pts) have historically been under-represented. In this study, we examine clinico-genomic data from a network of community oncology clinics to evaluate real-world mutational frequencies in Black, White, and Asian pts with cancer. Methods: We utilize Genospace, a precision medicine software, to harmonize clinical data from the Sarah Cannon research network of U.S. community oncology practices with genomic data from commercial NGS vendors. Pts with known race and NGS test results from September 2015 to November 2021 are assessed. Variants of uncertain significance (called by NGS vendor) are excluded. Asian pts are not evaluable in all instances because of low pt number. Statistical analyses include Chi squared and two sample test of proportions with Benjamini-Hochberg false discovery rate correction. Results: A total of 18,399 pts are assessed, with 7% Black (n = 1,319), 92% White (n = 16,903), and 1% Asian (n = 177) pt representation. The most common tumor types include non-small-cell lung (NSCLC; 27% of Black pts, 30% of White pts, 33% of Asian pts), colorectal (15% of Black pts, 12% of White pts, 14% of Asian pts), breast (14% of Black pts, 11% of White pts, 9% of Asian pts), and prostate (6% of Black pts, 5% of White pts, 3% of Asian pts) at roughly equal proportions across racial groups. In NSCLC, EGFR mutations and ALK fusions significantly enrich in Asian pts while KRAS G12C is detected more frequently in Black patients (see table for details; Black: n = 353, White: n = 5,127, Asian: n = 58). In both prostate and breast cancers, plasma-based NGS is less common in Black pts than White pts (plasma-to-tissue ratio = 1.1 (Black prostate: n = 82); 1.5 (White prostate: n = 863); 0.6 (Black breast: n = 178); 0.9 (White breast: n = 1,918)). In prostate, AR mutations are detected less frequently in Black pts. However, when considering plasma-NGS results alone the detection frequency is roughly equal. Similarly, the gap in ESR1 detection frequencies in breast decreases when considering plasma-NGS results alone. Conclusions: Actionable alterations are detected across racial groups at variable frequencies with potential biological and technical underpinnings. In scenarios where plasma-NGS is commonly used to monitor for resistance mutations, discrepancies in NGS ordering patterns likely affect detection frequencies. [Table: see text]

Cancer-related somatic mutations alter adenosine A1 receptor pharmacology-A focus on mutations in the loops and C-terminus.

G protein-coupled receptors (GPCRs) are known to be involved in tumor progression and metastasis. The adenosine A1 receptor (A1 AR) has been detected to be over-expressed in various cancer cell lines. However, the role of A1 AR in tumor development is not yet well characterized. A series of A1 AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. In this study, we have investigated the pharmacology of mutations located outside of the 7-transmembrane domain by using a "single-GPCR-one-G protein" yeast system. Concentration-growth curves were obtained with the full agonist CPA for 12mutant receptors and compared to the wild-type hA1 AR. Most mutations located at the extracellular loops (EL) reduced the levels of constitutive activity of the receptor and agonist potency. For mutants at the intracellular loops (ILs) of the receptor, an increased constitutive activity was found for mutant receptor L211R5.69 , while a decreased constitutive activity and agonist response were found for mutant receptor L113F34.51 . Lastly, mutations identified on the C-terminus did not significantly influence the pharmacological function of the receptor. A selection of mutations was also investigated in a mammalian system. Overall, similar effects on receptor activation compared to the yeast system were found with mutations located at the EL, but some contradictory effects were observed for mutations located at the IL. Taken together, this study will enrich the insight of A1 AR structure and function, enlightening the consequences of these mutations in cancer. Ultimately, this may provide potential precision medicine in cancer treatment.

Double-layer omics analysis of castration- and X-ray-resistant prostate cancer cells.

Castration-resistant prostate cancer shows resistance to not only androgen deprivation therapy (ADT) but also X-ray therapy. On the other hand, carbon ion beams have a high biological effect and are used for various cancers showing resistance to X-ray therapy. The purposes of this study are to clarify the difference in the sensitivity of Castration-resistant prostate cancer to X-ray and carbon ion beams and to elucidate the mechanism. The androgen-insensitive prostate cancer cell line LNCaP-LA established by culturing the androgen-sensitive prostate cancer cell line LNCaP for 2 years in androgen-free medium was used for this study. First, colony formation assays were performed to investigate its sensitivity to X-ray and carbon ion beams. Next, DNA mutation analysis on 409 cancer-related genes and comprehensive transcriptome analysis (RNA-seq) were performed with a next-generation sequencer. Lethal dose 50 values of X-rays for LNCaP and LNCaP-LA were 1.4 Gy and 2.8 Gy, respectively (P < 0.01). The Lethal dose 50 values of carbon ion beams were 0.9 Gy and 0.7 Gy, respectively (P = 0.09). On DNA mutation analysis, AR mutation was observed specifically in LNCaP-LA. From RNA-seq, 181 genes were identified as differentially expressed genes (DEGs; FDR <0.10, P < 0.00076) between LNCaP and LNCaP-LA. Function analysis suggested that cell death was suppressed in LNCaP-LA, and pathway analysis suggested that the NRF2-pathway involved in intracellular oxidative stress prevention was activated in LNCaP-LA. LNCaP-LA showed X-ray resistance compared to LNCaP and sensitivity to carbon ion beams. The AR mutation and the NRF2-pathway were suggested as causes of resistance.

Germline BRCA2, ATM and CHEK2 alterations shape somatic mutation landscapes in prostate cancer.

148 Background: Prostate cancer (PCa) is a highly heritable malignancy. Germline mutations in homologous recombination repair (HRR) genes, especially in in ATM, BRCA2 and CHEK2, are common in men with PCa, but how these influence somatic mutations is unknown. Characterizing the somatic landscape of tumors from germline HRR-altered PCa patients may provide insights into further therapeutic strategies and identify individuals most likely to benefit from existing therapies. Methods: We retrospectively identified 118 men with germline HRR-altered PCa from Johns Hopkins with available somatic mutation data (g ATM, n = 30; g BRCA2, n = 58; g CHEK2, n = 30). In these patients, somatic NGS analysis were done primarily using prostatic (rather than metastatic) tissues. The proportion of patients in each group with somatic biallelic inactivation was determined. We then compared the somatic mutation landscapes of the 3 groups, focusing on key PCa-relevant genes including AR, TMPRSS2-ERG, PTEN, TP53, BRCA2, ATM, CDK12, APC, FOXA1, AKT1, PIK3CA, and KDM6A among others. Somatic mutation data from primary prostate cancer specimens (n = 3965) found in pooled studies (n = 23) from cBioPortal were included as a reference standard for comparison. Results: Biallelic somatic inactivation was detected in 59% (34/58) of g BRCA2-altered, 47% (14/30) of g ATM-altered, and 0% (0/30) of g CHEK2-altered patients. In all 118 germline HRR-altered individuals, the most common somatic mutations were in BRCA2 (31%), FOXA1 (26%), TMPRSS2-ERG (20%), TP53 (16%), PTEN (15%), and ATM (15%). g BRCA2-altered patients had a higher prevalence of somatic mutations in AR (9% vs 3% in cBioPortal, P &lt; 0.05) and FOXA1 (29% vs 13% in cBioPortal, P &gt; 0.05), suggesting a greater dependency on AR signaling, but a lower prevalence of TMPRSS2-ERG fusions (17% vs 33% in cBioPortal, P &lt; 0.05). g ATM-altered patients had a strikingly low prevalence of somatic TP53 mutations (3% vs 25% in cBioPortal, P &lt; 0.01), indicating mutual exclusivity between these genes. g CHEK2-altered patients had a higher prevalence of somatic CDK12 mutations (10% vs 0% in gATM/gBRCA2 patients, P &lt; 0.05), implying an interaction between CHEK2 and CDK12. No differences were seen between the three groups with respect to alterations in the PI3K/PTEN pathway, WNT pathway, or chromatin-related genes. Conclusions: Biallelic inactivation was frequently observed in PCa patients harboring g ATM and g BRCA2 mutations, but not in those with g CHEK2 mutations. g BRCA2-altered patients were enriched for somatic AR (and FOXA1) mutations and were depleted for TMPRSS2-ERG fusions. g ATM-altered patients were depleted for somatic TP53 mutations, while g CHEK2-altered patients were enriched for somatic CDK12 mutations. Thus, germline mutations appear to impose selective pressures that influence somatic mutation landscapes in PCa.

Circulating tumor DNA responses to high-dose testosterone injections in CRPC patients.

173 Background: Some proportion of men with CRPC have favorable responses to high doses of testosterone (HDT) and some do not. Genomic determinants of these responders and non-responders are poorly understood. Herein ctDNA predictors of response are assessed using a responder/non-responder analysis. Methods: All men with CRPC had been pretreated with abiraterone, enzalutamide, or both. A ctDNA test was obtained prior to high doses of testosterone (administered as a dose of 400 mg IM testosterone cypionate every 3-4 weeks). Guardant 360 was used to analyze ctDNA (with an allelic fraction cut-off of &gt; 0.5%) to ascertain the presence or absence of pathogenic mutations. Lower allelic fractions were not analyzed. Responders had a PSA decline of 50% or more (N = 16), non-responders received at least two doses of testosterone but never had any PSA decline at all (N = 20). All patients were treated at Tulane Cancer Center. Results: AR amplifications were more commonly detected (p = 0.036) pre-treatment in the ctDNA of responders (5/16) as compared to non-responders (1/20). No differences were found in those with common AR mutations; T878A was detected in 2/16 responders and 2/20 non-responders, L702H was detected in 1/16 responders and 2/20 non-responders. No differences were seen with regard to TP53 mutations, 6/20 non-responders and 7/16 responders. Non- AR/non- TP53 mutations were not distinct in the two groups but trended (P = 0.15) toward being more common in non-responders (5/16 in responders versus 11/20 non-responders). Pre- and post- ctDNA analyses were conducted in 5/6 patients for those with AR amplification at baseline. In all 5 of these patients, the degree of AR amplification diminished after testosterone injections. Conclusions: In this analysis of CRPC patients who were responders and non-responders to 400 mg testosterone cypionate q 3-4 weeks (post-abiraterone and/or enzalutamide), only AR amplifications in ctDNA were predictive of response. In all measured patients, the degree of AR amplification in the ctDNA diminished after testosterone injections.

Novel mutation signatures in the prognosis of EGFR-TKIs targeted therapy for non-small cell lung cancer patients based on the 1000-gene panel sequencing.

The application of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) may be affected by somatic mutations. The purpose of this study was to explore the effect of mutations on the prognosis and tumor markers of NSCLC patients treated with EGFR-TKIs. 21 NSCLC patients treated with EGFR-TKIs were selected, and the targeted sequencing of the tumor tissues or whole blood samples with the 1000-gene panel was conducted to screen mutations. Afterward, functional enrichment analysis was performed based on mutant genes. Subsequently, the correlation between mutations and clinical indicators, prognosis, and tumor markers were analyzed. Finally, the prognosis after taking osimertinib was compared between NSCLC patients with EGFR p.T790M positive and negative mutations, and the EGFR p.T790M concomitant and uncommon mutations were screened. A total of 485 mutations in 251 genes were identified, in which MTOR, AXIN2, AR, EGFR, NOTCH1, and HRAS mutations were significantly correlated with PFS and/or tumor markers. There was no significant difference in PFS, therapeutic effect, and prognosis between EGFR p.T790M positive and negative patients who received osimertinib treatment. Besides, we also found 80 concomitant mutations and 54 uncommon mutations of EGFR p.T790M. AR, HRAS, EGFR, AXIN2, NOTCH1, and MTOR might be key genes to the prognosis of NSCLC treated with EGFR-TKIs. Osimertinib has certain efficacy in EGFR p.T790M negative NSCLC patients.

Case Report: Molecular Characterization of Aggressive Malignant Retroperitoneal Solitary Fibrous Tumor: A Case Study

Solitary fibrous tumors (SFT) are mesenchymal neoplasms with a favorable prognosis usually originating from the visceral pleura. Rarely, they may occur at various extrapleural sites and show malignant behavior coupled with dedifferentiation. NAB2-STAT6 fusion gene and STAT6 nuclear expression are biomarkers for diagnosis of SFT in addition to CD34, Bcl-2, and CD99. Furthermore, several reports have shown specific NAB2-STAT6 fusion variants and loss of STAT6 protein expression are associated with malignancy. We report a rare case of retroperitoneal SFT which rapidly progressed to death within 35 days after admission. Autopsy found a primary tumor containing both benign and malignant histologies, with multiple metastatic sites similar to the malignant, dedifferentiated tumor. STAT6 was detected in the primary differentiated tumor but not in the primary dedifferentiated tumor or lung/liver metastases. However, the NAB2-STAT6 fusion gene (NAB2ex6/STAT6ex16 variant) was detected in the primary tumor and lung/liver metastases. Intriguingly, fusion gene expression at the transcriptional level was downregulated in the dedifferentiated tumors compared to the differentiated tumor. We further performed target DNA sequencing and found gene mutations in TP53, FLT3, and AR in the dedifferentiated tumors, with TP53 mutations especially found among them. We demonstrate that downregulation of NAB2-STAT6 fusion gene at the transcriptional level is associated with malignant SFT for the first time. Moreover, the present study supports the idea that TP53 mutations promote malignancy in SFTs.

Somatic driver mutation prevalence in 1844 prostate cancers identifies ZNRF3 loss as a predictor of metastatic relapse

Driver gene mutations that are more prevalent in metastatic, castration-resistant prostate cancer (mCRPC) than localized disease represent candidate prognostic biomarkers. We analyze 1,844 localized (1,289) or mCRPC (555) tumors and quantify the prevalence of 113 somatic driver single nucleotide variants (SNVs), copy number aberrations (CNAs), and structural variants (SVs) in each state. One-third are significantly more prevalent in mCRPC than expected while a quarter are less prevalent. Mutations in AR and its enhancer are more prevalent in mCRPC, as are those in TP53, MYC, ZNRF3 and PRKDC. ZNRF3 loss is associated with decreased ZNRF3 mRNA abundance, WNT, cell cycle & PRC1/2 activity, and genomic instability. ZNRF3 loss, RNA downregulation and hypermethylation are prognostic of metastasis and overall survival, independent of clinical and pathologic indices. These data demonstrate a strategy for identifying biomarkers of localized cancer aggression, with ZNRF3 loss as a predictor of metastasis in prostate cancer.

Discovery of ARD-2585 as an Exceptionally Potent and Orally Active PROTAC Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report herein the discovery of exceptionally potent and orally bioavailable PROTAC AR degraders with ARD-2585 being the most promising compound. ARD-2585 achieves DC50 values of ≤0.1 nM in the VCaP cell line with AR gene amplification and in the LNCaP cell line carrying an AR mutation. It potently inhibits cell growth with IC50 values of 1.5 and 16.2 nM in the VCaP and LNCaP cell lines, respectively, and achieves excellent pharmacokinetics and 51% of oral bioavailability in mice. It is more efficacious than enzalutamide in inhibition of VCaP tumor growth and does not cause any sign of toxicity in mice. ARD-2585 is a promising AR degrader for extensive investigations for the treatment of advanced prostate cancer.

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Abstract 863: Circadian gene expression in metastatic sites and association with survival in metastatic castration-resistant prostate cancer

Abstract Introduction: In experimental and epidemiological studies, alterations in several core circadian genes at the germline and tumor level have been associated with prostate cancer. The aim of this study was to investigate mRNA expression of circadian related genes in men with metastatic castration-resistant prostate cancer (mCRPC), and the association with survival. Methods: We assessed whole exome and RNA sequencing data from 317 mCRPC patients from the Stand Up to Cancer-Prostate Cancer Foundation (SU2C-PCF) database. Data were obtained from six sites: metastasis to bone (n=107; n=65 deaths), lymph node (n=129; n=88 deaths), liver (n=42; n=35 deaths), lung (n=6; n=3 deaths), and other soft tissue (n=26; n=20 deaths), as well as primary prostate (n=7; n=5 deaths) over a median follow-up of 71.8 months. We evaluated expression of twelve core circadian genes (ARNTL, CLOCK, CRY1, CRY2, CSNK1E, NR1D1, NPAS2, PER1, PER2, PER3, RORA, TIMELESS) as transcripts per million (TPM). We used the correlation of correlations method to estimate inter-gene correlations between tissue. Unpaired Wilcoxon rank sum tests compared circadian expression differences with tumor mutations in AR and p53, two of the most common genomic alterations in mCRPC. We conducted multivariable Cox regression, overall and stratified by tissue type, to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs) for expression (modeled continuously) and overall survival, adjusted for age and PSA at diagnosis, Gleason, treatment, and histology. Results: Many genes showed low or negative correlation across tissues, with the greatest discordance in CSNK1E (μICC=0.10), and greatest concordance in TIMELESS (μICC=0.55). Lower expression of ARNTL was found in patients with alterations to both p53 and AR. Similarly, higher expression of PER2 and RORA was found in AR-/p53+, compared to those AR+ and AR-/p53-. Higher expression of TIMELESS was associated with risk of death overall and across all tissue sites (HRoverall: 1.02, 95% CI: 1.01-1.03). In liver, higher expression of CLOCK (HR: 0.22, 95% CI: 0.07 - 0.71) and CSNK1E (HR: 0.87, 95% CI: 0.76 - 1.00), and lower expression of CRY1 (HR: 1.62, 95% CI: 1.16 - 2.26) was associated with a lower risk of death. Higher expression of CRY2 (HR: 1.25, 95% CI: 1.02 - 1.53) in liver, but lower expression in bone (HR: 0.95, 95% CI: 0.90 - 1.00) was associated with an increased risk of death. We found no association between ARNTL, NR1D1, NPAS2, PER3, or RORA and survival in any metastatic site. Conclusion: Our results show that circadian gene expression is altered in tissue from mCRPC patients, with substantial heterogeneity in circadian related expression patterns between metastatic tissue sites. These results support prior research on the role of circadian gene expression, particularly CRY1 and CLOCK, and outcomes in localized prostate cancer. Citation Format: Benjamin D. Booker, Konrad H. Stopsack, Travis A. Gerke, Kathryn Penny, Philip W. Kantoff, Lorelei A. Mucci, Sarah C. Markt, PCF/SU2C International Prostate Cancer Dream Team. Circadian gene expression in metastatic sites and association with survival in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 863.

Abstract 549: Genomic characterization and monitoring molecular response to treatment in African American (AA) advanced prostate cancer (PC) patients (pts) via next-generation sequencing (NGS): Real-world experience in a safety net hospital Oncology clinic

Abstract Background: AA men have higher PC incidence and mortality than white men. Healthcare access and other socioeconomic factors contribute to these disparities. The high cost of NGS can become an additional factor for disparity. Indeed, minority and uninsured pts were underrepresented in prior NGS studies and NGS-based Precision Oncology clinical trials. Methods: Ben Taub Hospital (BTH) is a safety net hospital serving a racially/ethnically diverse pt population (91% minorities). We retrospectively analyzed NGS data obtained via Tempus|xT tissue assay (DNA sequencing of 648 genes in tumor tissue at 500x depth) and/or Tempus|xF liquid biopsy assay (ctDNA sequencing of 105 genes at 5,000x depth) for germline and/or somatic mutations detected in 117 BTH pts [61 self-identified AA and 56 white (including white Hispanic)] receiving treatment for locally advanced, biochemically recurrent or metastatic PC. We also analyzed de-identified NGS data from a nationwide cohort of 2090 metastatic PC pts (225 AA) previously sequenced with xT and/or xF by Tempus Labs (Chicago, IL). Results: AA BTH PC pts exhibited high frequencies of BRCA2 (14.8%), SPOP (19.7%), AR (16.4%) and TP53 (49.1%) mutations (respective frequencies in white BTH pts were 9%, 7%, 3.6% and 12.5%). TMPRSS2 fusions were less frequent in AA than white pts. Upon treatment with active therapy (hormonal or taxane), the allele frequency of PC-derived ctDNA mutations in the liquid biopsy declined significantly and concordantly to the biochemical (PSA) response. In addition, two out of 61 (3.3%) AA BTH PCs had microsatellite instability [a frequency comparable to that seen in white BTH PC pts (3.6%) and in the nationwide Tempus Labs cohort (3.6% of AA PC pts)], and experienced prompt and durable clinical, biochemical and molecular responses to pembrolizumab, including disappearance of PC-derived mutant ctDNA. On the other hand, four AA PC pts who had progressed on abiraterone or enzalutamide with ctDNA findings of AR mutations previously reported as sensitive in vitro to the newest FDA-approved AR antagonist darolutamide, did not achieve clinical or biochemical response on darolutamide treatment and that was paralleled by lack of molecular (ctDNA fraction) response. Conclusions: The high mutation frequency in key PC drivers in AA pts at our safety net hospital can be attributed to underlying disease biology or the more advanced disease at presentation in AA pts with socioeconomic factors delaying access to healthcare. Wide use of NGS testing is necessary to improve early access of underserved minority populations to novel targeted therapies and to biomarker-based Precision Oncology clinical trials. Liquid biopsy is a minimally invasive tool that allows longitudinal monitoring of response (or lack thereof) to treatment. Citation Format: Tamer Khashab, Salma Kaochar, Alexander D. Le, Samantha Cohen, Attiya B. Noor, Heidi Dowst, Neda Zarrin-Khameh, Michael A. Brooks, Guilherme Godoy, Maria A. Berezina, Anna E. Schwarzbach, Michael E. Scheurer, Martha P. Mims, Nicholas Mitsiades. Genomic characterization and monitoring molecular response to treatment in African American (AA) advanced prostate cancer (PC) patients (pts) via next-generation sequencing (NGS): Real-world experience in a safety net hospital Oncology clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 549.

Comprehensive molecular profiling of advanced cancers in a real-world setting using an ultrasensitive amplicon-based next-generation sequencing (NGS) liquid biopsy assay.

3062 Background: Molecular profiling of circulating tumor DNA (ctDNA) by NGS has demonstrated the value of liquid biopsy in informing treatment decisions and monitoring disease progression in cancer patients. Here we report results from the real-world clinical application of an ultrasensitive amplicon-based NGS liquid biopsy assay for advanced cancers. Methods: Plasma cell-free DNA (cfDNA) from 1,338 consecutive samples (51.3% lung, 15.6% breast, 8.4% colorectal, 26.8% from 18 other cancer types; 86.3% of cancer samples were metastatic) underwent real-world testing in a Singapore-based, CAP-accredited, CLIA-certified laboratory from Jan 2018 – Nov 2020. Genomic alterations were analyzed using an amplicon-based NGS assay that detects alterations in 80 cancer-related genes, microsatellite instability (MSI) and cancer-causing viruses, with previously validated cfDNA detection limits of 0.1% variant allele frequency (VAF) for SNPs and indels, 5% tumor fraction, and 2 IU/mL plasma, respectively. Results: ctDNA was successfully detected in 70.0% of cancer samples (76.3% from metastatic and 24.7% from localized tumors) for a total of 977 unique variants. Across ctDNA-positive samples the median VAF was 1.4%, and 8.5% of all reported variants had VAFs between 0.01 – 0.09%. The most frequently altered genes were TP53 (48.8%), EGFR (36.9%), KRAS (17.4%), PIK3CA (15.3%), and APC (9.5%). In ctDNA-positive lung cancer, 74.6% of samples (66.5% EGFR+) harbored ≥1 actionable target, with 29.9% of patients treated with a first- or second-generation EGFR tyrosine kinase inhibitor harboring an EGFR T790M resistance mutation. Among breast cancers, ESR1 or PIK3CA alterations predicting drug resistance/response were detected in 44.2% of HR+ cases. In colorectal cancer, alterations implicated in anti-EGFR resistance ( NRAS, KRAS, BRAF, ERBB2) were found in 50.0% of samples. Profiling of cfDNA also enabled cancer monitoring, even among cancers with low genetic biomarker prevalence. 50% of nasopharyngeal cancer samples were Epstein-Barr virus-positive while 35.7% of liver cancer samples were Hepatitis B virus-positive, enabling an additional yield of 14.3% and 7.1% respectively, over ctDNA detection. cfDNA profiling of cancers of unknown primary origins informed potential tumor origins via the detection of tumor type-specific alterations such as mutations in AR, ESR1, GNAS, EGFR genes, viral biomarkers, and MSI. Conclusions: The use of a comprehensive pan-cancer liquid biopsy panel with rational inclusion of actionable and driver genes resulted in detection of ctDNA in the vast majority of clinical cases processed routinely. These findings support the utility of ultrasensitive amplicon-based NGS liquid biopsy assays in probing cancer drug sensitivity/resistance, tumor burden monitoring, and molecular characterization of tumors.