Next-generation sequencing of prostate cancer: genomic and pathway alterations,potential actionability patterns,and relative rate of use of clinical-grade testing

Despite being one of the most common cancers, treatment options for prostate cancer are limited. Novel approaches for advanced disease are needed. We evaluated the relative rate of use of clinical-grade next generation sequencing (NGS) in prostate cancer, as well as genomic alterations identified and their potential actionability. Of 4864 patients from multiple institutions for whom NGS was ordered by physicians, only 67 (1.4%) had prostate cancer, representing 1/10 the ordering rate for lung cancer. Prostate cancers harbored 148 unique alterations affecting 63 distinct genes. No two patients had an identical molecular portfolio. The median number of characterized genomic alterations per patient was 3 (range, 1 to 9). Fifty-six of 67 patients (84%) had ≥ 1 potentially actionable alteration. TMPRSS2 fusions affected 28.4% of patients. Genomic aberrations were most frequently detected in TP53 (55.2% of patients), PTEN (29.9%), MYC (17.9%), PIK3CA (13.4%), APC (9.0%), BRCA2 (9.0%), CCND1 (9.0%), and RB1 genes (9.0%). The PI3K (52.2% of patients), WNT (13.5%), DNA repair (17.9%), cell cycle (19.4%), and MAPK (14.9%) machinery were commonly impacted. A minority of patients harbored BRAF, NTRK, ERBB2, or mismatch repair gene abnormalities, which are highly druggable in some cancers. Only ~ 10% of prostate cancer trials (clinicaltrials.gov, year 2017) applied a (non-hormone) biomarker before intervention. In conclusion, though use of clinical-grade NGS is relatively low and only a minority of trials deploy DNA-based biomarkers, many prostate cancer-associated molecular alterations may be pharmacologically tractable with genomcially targeted therapy or, in the case of mismatch repair anomalies, with checkpoint inhibitor immunotherapy.     

Evolving treatment paradigms for locally advanced and metastatic prostate cancer

While men with early stage prostate cancer typically enjoy long-term survival after definitive management, for those who present with locally advanced or metastatic disease, survival is compromised. Multimodality therapy can prolong survival in these patients, with state-of-the-art options including intensity-modulated radiation or brachytherapy in conjunction with androgen ablation, adjuvant androgen ablation and/or chemotherapy with radical retropubic prostatectomy. In addition, novel biological therapies are being explored to target the unique molecular changes in prostate cancer cells and their interactions with the microenvironment. With these advances the outlook will undoubtedly improve, even for patients presenting with advanced disease. Careful application of these emerging therapies to a select group of prostate cancer patients most likely to obtain benefit from them is the challenge for urologists, medical oncologists and radiation oncologists for the future.     

Clinical utility of comprehensive genomic profiling tests for advanced or metastatic solid tumor in clinical practice

Previous clinical trials indicate that 10%–25% of patients received genomically matched therapy after comprehensive genomic profiling (CGP) tests. However, the clinical utility of CGP tests has not been assessed in clinical practice. We assessed the clinical utility of CGP tests for advanced or metastatic solid tumor and determined the proportion of patients receiving genomically matched therapy among those with common and non‐common cancers. From August 2019 to July 2020, a total of 418 patients had undergone CGP tests, and the results were discussed through the molecular tumor board at our site. The median age of patients was 57 (range: 3–86) years. Colorectal cancer was the most common, with 47 (11%) patients. Actionable genomic alterations (median 3, range: 1–17) were identified in 368 (88.0%) of 418 patients. Druggable genomic alterations were determined in 196 (46.9%) of 418 patients through the molecular tumor board. Genomically matched therapy was administered as the subsequent line of therapy in 51 (12.2%) patients, which is comparable to the proportion we previously reported in a clinical trial (13.4%) (p = 0.6919). The proportion of patients receiving genomically matched therapy was significantly higher among those with common cancers (16.2%) than non‐common cancers (9.4%) (p = 0.0365). Genomically matched therapy after the CGP tests was administered to 12.2% of patients, which is similar to the proportion reported in the previous clinical trials. The clinical utility of CGP tests in patients with common cancers greatly exceeded that in patients with non‐common cancers.     

Expert panel consensus recommendations on the use of circulating tumor DNA assays for patients with advanced solid tumors

Comprehensive genomic profiling is increasingly used to facilitate precision oncology based on molecular stratification. In addition to conventional tissue comprehensive genomic profiling, comprehensive genomic profiling of circulating tumor DNA has become widely utilized in cancer care owing on its advantages, including less invasiveness, rapid turnaround time, and capturing heterogeneity. However, circulating tumor DNA comprehensive genomic profiling has some limitations, mainly false negatives due to low levels of plasma circulating tumor deoxyribonucleic acid and false positives caused by clonal hematopoiesis. Nevertheless, no guidelines and recommendations fully address these issues. Here, an expert panel committee involving representatives from 12 Designated Core Hospitals for Cancer Genomic Medicine in Japan was organized to develop expert consensus recommendations for the use of circulating tumor deoxyribonucleic acid‐based comprehensive genomic profiling. The aim was to generate guidelines for clinicians and allied healthcare professionals on the optimal use of the circulating tumor DNA assays in advanced solid tumors and to aid the design of future clinical trials that utilize and develop circulating tumor DNA assays to refine precision oncology. Fourteen clinical questions regarding circulating tumor deoxyribonucleic acid comprehensive genomic profiling including the timing of testing and considerations for interpreting results were established by searching and curating associated literatures, and corresponding recommendations were prepared based on the literature for each clinical question. Final consensus recommendations were developed by voting to determine the level of each recommendation by the Committee members.     

Somatic mutations reveal complex metastatic seeding from multifocal primary prostate cancer

Primary prostate cancer shows a striking intraorgan molecular heterogeneity, with multiple spatially separated malignant foci in the majority of patients. Metastatic prostate cancer, however, typically reveals more homogenous molecular profiles, suggesting a monoclonal origin of the metastatic lesions. Longitudinal mutational spectra, comparing multiple primary lesions with metastases from the same patients remain poorly defined. We have here analyzed somatic mutations in multisampled, spatio-temporal biobanked lesions (38 samples from primary foci and 1 sample from each of 8 metastases from seven prostate cancer patients) applying a custom-designed panel targeting 68 prostate cancer relevant genes. The metastatic samples were taken at time of primary surgery and up to 7 years later, and sampling included circulating tumor DNA in plasma or solid metastatic tissue samples. A total of 282 somatic mutations were detected, with a range of 0 to 25 mutations per sample. Although seven samples had solely private mutations, the remaining 39 samples had both private and shared mutations. Seventy-four percent of mutations in metastases were not found in any primary samples, and vice versa, 96% of mutations in primary cancers were not found in any metastatic samples. However, for three patients, shared mutations were found suggesting the focus of origin, including mutations in AKT1, FOXA1, HOXB13, RB1 and TP53. In conclusion, the spatio-temporal heterogeneous nature of multifocal disease is emphasized in our study, and underlines the importance of testing a recent sample in genomics-based precision medicine for metastatic prostate cancer.     

Comprehensive genomic profiling of lung cancer using a validated panel to explore therapeutic targets in East Asian patients

People of East Asian ethnicity have a different prevalence of and show unique clinical characteristics and tumor histology of oncogenic mutations. However, only limited studies have explored the landscape of genomic alterations in lung adenocarcinoma derived from Asian patients thus far. In this single‐center study, with an aim to elucidate the mutational profile of lung cancer in people of Chinese ethnicity and to use the obtained information to guide decision‐making for treatment, we employed a well‐validated assay to perform comprehensive genomic characterization of tumor specimens from 306 Chinese lung cancer patients. A total of 845 individual genomic alterations were found in 145 tumor‐related genes with a median of 2.8 alterations (range: 1–18) per sample. The most frequently mutated genes were EGFR (46.7%), TP53 (21.2%), ALK (12.1%; 8.8% of mutation and 3.3% of rearrangement) and KRAS (10.1%). Upon comparison with the Cancer Genome Atlas dataset, we found that EGFR was mutated at a much higher frequency in our cohort than in Caucasians, whereas KRAS was only found in 10.1% of our Chinese patients. Clinically relevant genomic alterations were identified in 185 (60.5%) patients, including 50% in adenocarcinoma patients and 14% in squamous cell carcinoma patients. Our findings suggest that the Asian ethnicity is significantly different from the Caucasian ethnicity with regard to the presence of somatic driver mutations. Furthermore, we showed that the use of a comprehensive genotyping approach could help identify actionable genomic alterations that have potential impact on therapeutic decisions.     

Genomic alterations in plasma DNA from patients with metastasized prostate cancer receiving abiraterone or enzalutamide

In patients with metastatic castrate‐resistant prostate cancer (mCRPC), circulating tumor DNA (ctDNA) analysis offers novel opportunities for the development of non‐invasive biomarkers informative of treatment response with novel agents targeting the androgen‐receptor (AR) pathway, such as abiraterone or enzalutamide. However, the relationship between ctDNA abundance, detectable somatic genomic alterations and clinical progression of mCRPC remains unexplored. Our study aimed to investigate changes in plasma DNA during disease progression and their associations with clinical variables in mCRPC patients. We analyzed ctDNA in two cohorts including 94 plasma samples from 25 treatment courses (23 patients) and 334 plasma samples from 125 patients, respectively. We conducted whole‐genome sequencing (plasma‐Seq) for genome‐wide profiling of somatic copy number alterations and targeted sequencing of 31 prostate cancer‐associated genes. The combination of plasma‐Seq with targeted AR analyses identified prostate cancer‐related genomic alterations in 16 of 25 (64%) treatment courses in the first cohort, in which we demonstrated that AR amplification does not always correlate with poor abiraterone and enzalutamide therapy outcome. As we observed a wide variability of ctDNA levels, we evaluated ctDNA levels and their association with clinical parameters and included the second, larger cohort for these analyses. Employing altogether 428 longitudinal plasma samples from 148 patients, we identified the presence of bone metastases, increased lactate dehydrogenase and prostate‐specific antigen (PSA) as having the strongest association with high ctDNA levels. In summary, ctDNA alterations are observable in the majority of patients with mCRPC and may eventually be useful to guide clinical decision‐making in this setting.     

Circulating tumor DNA in advanced solid tumors: Clinical relevance and future directions

The application of genomic profiling assays using plasma circulating tumor DNA (ctDNA) is rapidly evolving in the management of patients with advanced solid tumors. Diverse plasma ctDNA technologies in both commercial and academic laboratories are in routine or emerging use. The increasing integration of such testing to inform treatment decision making by oncology clinicians has complexities and challenges but holds significant potential to substantially improve patient outcomes. In this review, the authors discuss the current role of plasma ctDNA assays in oncology care and provide an overview of ongoing research that may inform real-world clinical applications in the near future.     

Single tube liquid biopsy for advanced non-small cell lung cancer

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM^high circulating tumor cells (CTC), EpCAM^low CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM^high CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM^low CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAM^high CTC, 15% had ≥2 EpCAM^low CTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM^high CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM^low CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.     

液体活检在非小细胞肺癌靶向治疗中应用

目的液体活检技术在肿瘤的检测方法中是尤为重要的检测手段。其在指导非小细胞肺癌(non-small cell lung cancer,NSCLC)靶向药物的选择、耐药监测以及预后评估等方面具有重要作用,本研究分析近年来国内外液体活检在NSCLC靶向治疗中的应用,以期明确液体活检对NSCLC靶向治疗的指导作用,进而有助于指导靶向药物在NSCLC治疗中的应用。方法应用PubMed、中国知网及中国期刊全文数据库检索系统,以"液体活检、循环肿瘤DNA、循环肿瘤细胞、靶向治疗、非小细胞肺癌"为关键词,检索2009-2019年发表的相关文献。纳入标准:(1)液体活检的机制;(2)靶向治疗在NSCLC中的应用;(3)NSCLC的治疗。排除标准:(1)中文非核心期刊的文献和英文非SCI收录文献;(2)结果重复且相对陈旧的实验研究。根据纳入标准和排除标准,最终29篇文献纳入分析。结果液体活检技术可对NSCLC的诊断、治疗及预后评估进行实时动态监测,及时获取肿瘤基因突变信息,在指导靶向药物选择,对耐药监测以及预后评估等方面具有重要作用。结论液体活检对指导NSCLC患者的靶向治疗具有重要作用,有助于NSCLC患者靶向药物治疗的选择。     

Comprehensive genomic profiling for patients with chemotherapy‐naïve advanced cancer

Comprehensive genomic profiling (CGP) testing by next‐generation sequencing has been introduced into clinical practice as part of precision cancer medicine to select effective targeted therapies. However, whether CGP testing at the time of first‐line chemotherapy could be clinically useful is not clear. We conducted this single‐center, prospective, observational study to investigate the feasibility of CGP testing for chemotherapy‐naïve patients with stage III/IV gastrointestinal cancer, rare cancer, and cancer of unknown primary, using the FoundationOne^® companion diagnostic (F1CDx) assay. The primary outcome was the detection rate of at least one actionable/druggable cancer genomic alteration. Actionable/druggable cancer genomic alterations were determined by the F1CDx report. An institutional molecular tumor board determined the molecular‐based recommended therapies. A total of 197 patients were enrolled from October 2018 to June 2019. CGP success rate was 76.6% (151 of 197 patients), and median turnaround time was 19 days (range: 10‐329 days). Actionable and druggable cancer genomic alterations were reported in 145 (73.6%) and 124 (62.9%) patients, respectively. The highest detection rate of druggable genomic alterations in gastrointestinal cancers was 80% in colorectal cancer (48 of 60 patients). Molecular‐based recommended therapies were determined in 46 patients (23.4%). CGP testing would be a useful tool for the identification of a potentially effective first‐line chemotherapy.     

Detection and characterization of invasive circulating tumor cells derived from men with metastatic castration‐resistant prostate cancer

The Vitatex cell‐adhesion matrix (CAM) platform allows for isolation of invasive circulating tumor cells (iCTCs). Here we sought to determine the utility of prostate‐specific membrane antigen (PSMA) as a metastatic castration‐resistant prostate cancer (mCRPC) iCTC biomarker, to identify solitary cells and clusters of iCTCs expressing either epithelial, mesenchymal, or stem cell markers, and to explore the feasibility of iCTC epigenomic analysis. CTCs were isolated and enumerated simultaneously using the Vitatex and CellSearch platforms in 23 men with mCRPC. CAM‐avid iCTCs were identified as nucleated cells capable of CAM uptake, but without detectable expression of hematopoietic lineage (HL) markers including CD45. iCTCs were enumerated immunocytochemically (ICC) and by flow cytometry. Whole‐genome methylation status was determined for iCTCs using the Illumina HumanMethylation27 BeadChip. Thirty‐four samples were collected for iCTC analysis. A median of 27 (range 0–800) and 23 (range 2–390) iCTCs/mL were detected by ICC and flow, respectively. In a subset of 20 samples, a median of seven CTCs/mL (range 0–85) were detected by the CellSearch platform compared to 26 by the CAM platform. iCTC clusters were observed in 17% of samples. iCTCs expressing PSMA as well as markers of EMT and stemness were detectable. The iCTC methylation profile highly resembled mCRPC. More CTCs were recovered using the CAM platform than the CellSearch platform, and the CAM platform allowed for the detection of iCTC clusters, iCTCs expressing EMT and stem‐cell markers, and characterization of the iCTC methylome. Correlation with clinical data in future studies may yield further insight into the functional significance of these findings.     

A comparison of isolated circulating tumor cells and tissue biopsies using whole-genome sequencing in prostate cancer

Previous studies have demonstrated focal but limited molecular similarities between circulating tumor cells (CTCs) and biopsies using isolated genetic assays. We hypothesized that molecular similarity between CTCs and tissue exists at the single cell level when characterized by whole genome sequencing (WGS). By combining the NanoVelcro CTC Chip with laser capture microdissection (LCM), we developed a platform for single-CTC WGS. We performed this procedure on CTCs and tissue samples from a patient with advanced prostate cancer who had serial biopsies over the course of his clinical history. We achieved 30X depth and ≥ 95% coverage. Twenty-nine percent of the somatic single nucleotide variations (SSNVs) identified were founder mutations that were also identified in CTCs. In addition, 86% of the clonal mutations identified in CTCs could be traced back to either the primary or metastatic tumors. In this patient, we identified structural variations (SVs) including an intrachromosomal rearrangement in chr3 and an interchromosomal rearrangement between chr13 and chr15. These rearrangements were shared between tumor tissues and CTCs. At the same time, highly heterogeneous short structural variants were discovered in PTEN, RB1, and BRCA2 in all tumor and CTC samples. Using high-quality WGS on single-CTCs, we identified the shared genomic alterations between CTCs and tumor tissues. This approach yielded insight into the heterogeneity of the mutational landscape of SSNVs and SVs. It may be possible to use this approach to study heterogeneity and characterize the biological evolution of a cancer during the course of its natural history.     

Identification of genomic signatures in circulating tumor cells from breast cancer

Levels of circulating tumor cells (CTCs) in blood have prognostic value in early and metastatic breast cancer. CTCs also show varying degrees of concordance with molecular markers of primary tumors they originate from. It is expected that individual cells reflect the heterogeneity and evolution of tumor cells as they acquire new functions and differential responses to chemotherapy. However, a degree of commonality is also plausible, highlighting alterations that allow tumor cells to perform CTC‐defining activities such as invasion and intravasation. Using a matched tumor‐normal approach, we performed high‐resolution copy number profiling of CTCs from breast cancer to identify occult changes occurring during progression to metastasis. We identified a signature of recurrent gain in CTCs, consisting of 90 minimal common regions (MCRs) of copy number gain. These were predominantly found across chromosome 19 and were identified at low frequencies (3–4%) in 787 primary breast carcinomas examined. CTC genomic signatures clustered into two groups independent of subtype: a dormancy‐related signature with 16 MCRs (AKT2, PTEN, CADM2); and a tumor‐aggressiveness related signature with 358 MCRs (ANGPTL4, BSG, MIR‐373). There were two MCRs in common between the groups on 19q13 and 21q21, containing genes involved in resistance to anoikis, TGFβ‐signaling and metastasis (TFF3, LTBP4, NUMBL). Furthermore, a region harboring the ERBB2 gene was gained in a majority of patients. Regions 20q13 and 15q24 were associated with distant metastasis. The distinctiveness of CTC signatures highlights cell populations with different functional or metastatic potential. Such novel targets could help to specifically identify and block dissemination.     

Multigene model for predicting metastatic prostate cancer using circulating tumor cells by microfluidic magnetophoresis

We aimed to isolate circulating tumor cells (CTCs) using a microfluidic technique with a novel lateral magnetophoretic microseparator. Prostate cancer–specific gene expressions were evaluated using mRNA from the isolated CTCs. A CTC‐based multigene model was then developed for identifying advanced prostate cancer. Peripheral blood samples were obtained from five healthy donors and patients with localized prostate cancer (26 cases), metastatic hormone‐sensitive prostate cancer (mHSPC, 10 cases), and metastatic castration‐resistant prostate cancer (mCRPC, 28 cases). CTC recovery rate and purity (enriched CTCs/total cells) were evaluated according to cancer stage. The areas under the curves of the six gene expressions were used to evaluate whether multigene models could identify mHSPC or mCRPC. The number of CTCs and their purity increased at more advanced cancer stages. In mHSPC/mCRPC cases, the specimens had an average of 27.5 CTCs/mL blood, which was 4.2 × higher than the isolation rate for localized disease. The CTC purity increased from 2.1% for localized disease to 3.8% for mHSPC and 6.7% for mCRPC, with increased CTC expression of the genes encoding prostate‐specific antigen (PSA), prostate‐specific membrane antigen (PSMA), and cytokeratin 19 (KRT19). All disease stages exhibited expression of the genes encoding androgen receptor (AR) and epithelial cell adhesion molecule (EpCAM), although expression of the AR‐V7 variant was relatively rare. Relative to each gene alone, the multigene model had better accuracy for predicting advanced prostate cancer. Our lateral magnetophoretic microseparator can be used for identifying prostate cancer biomarkers. In addition, CTC‐based genetic signatures may guide the early diagnosis of advanced prostate cancer.