Discovery and Validation of a 15-Gene Prognostic Signature for Clear Cell Renal Cell Carcinoma.

PURPOSE: Develop and validate gene expression-based biomarker associated with recurrent disease to facilitate risk stratification of clear cell renal cell carcinoma (ccRCC). MATERIALS AND METHODS: We retrospectively identified 110 patients who underwent radical nephrectomy for ccRCC (discovery cohort). Patients who recurred were matched on the basis of grade/stage to patients without recurrence. Capture whole-transcriptome sequencing was performed on RNA isolated from archival tissue using the Illumina platform. We developed a gene-expression signature to predict recurrence-free survival/disease-free survival (DFS) using a 15-fold lasso and elastic-net regularized linear Cox model. We derived the 31-gene cell cycle progression (mxCCP) score using RNA-seq data for each patient. Kaplan-Meier (KM) curves and multivariable Cox proportional hazard testing were used to validate the independent prognostic impact of the gene-expression signature on DFS, disease-specific survival (DSS), and overall survival (OS) in two validation data sets (combined n = 761). RESULTS: After quality control, the discovery cohort comprised 50 patients with recurrence and 41 patients without, with a median follow-up of 26 and 36 months, respectively. We developed a 15-gene (15G) signature, which was independently associated with worse DFS and DSS (DFS: hazard ratio [HR], 11.08 [95% CI, 4.9 to 25.1]; DSS: HR, 9.67 [95% CI, 3.4 to 27.7]) in a multivariable model adjusting for clinicopathologic parameters (including stage, size, grade, and necrosis [SSIGN] score and Memorial Sloan Kettering Cancer Center nomogram) and mxCCP score. The 15G signature was also independently associated with worse DFS and DSS in both validation data sets (Validation A [n = 382], DFS: HR, 2.6 [95% CI, 1.6 to 4.3]; DSS: HR, 3 [95% CI, 1.4 to 6.1] and Validation B (n = 379), DFS: HR, 2.1 [95% CI, 1.2 to 3.6]; OS: HR, 3 [95% CI, 1.6 to 5.7]) adjusting for clinicopathologic variables and mxCCP score. CONCLUSION: We developed and validated a novel 15G prognostic signature to improve risk stratification of patients with ccRCC. Pending further validation, this signature has the potential to facilitate optimal treatment allocation.

Integrative multi-region molecular profiling of primary prostate cancer in men with synchronous lymph node metastasis.

Localized prostate cancer is frequently composed of multiple spatially distinct tumors with significant inter- and intra-tumoral molecular heterogeneity. This genomic diversity gives rise to many competing clones that may drive the biological trajectory of the disease. Previous large-scale sequencing efforts have focused on the evolutionary process in metastatic prostate cancer, revealing a potential clonal progression to castration resistance. However, the clonal origin of synchronous lymph node (LN) metastases in primary disease is still unknown. Here, we perform multi-region, targeted next generation sequencing and construct phylogenetic trees in men with prostate cancer with synchronous LN metastasis to better define the pathologic and molecular features of primary disease most likely to spread to the LNs. Collectively, we demonstrate that a combination of histopathologic and molecular factors, including tumor grade, presence of extra-prostatic extension, cellular morphology, and oncogenic genomic alterations are associated with synchronous LN metastasis.

Analysis of the Tumor Immune Microenvironment (TIME) in Clear Cell Renal Cell Carcinoma (ccRCC) Reveals an M0 Macrophage-Enriched Subtype: An Exploration of Prognostic and Biological Characteristics of This Immune Phenotype.

There is a need to optimize the treatment of clear cell renal cell carcinoma (ccRCC) patients at high recurrence risk after nephrectomy. We sought to elucidate the tumor immune microenvironment (TIME) of localized ccRCC and understand the prognostic and predictive characteristics of certain features. The discovery cohort was clinically localized patients in the TCGA-Kidney Renal Clear Cell Carcinoma (KIRC) project (n = 382). We identified an M0 macrophage-enriched cluster (n = 25) in the TCGA-KIRC cohort. This cluster's median progression-free survival (PFS) and overall survival (OS) were 40.4 and 45.3 months, respectively, but this was not reached in the others (p = 0.0003 and <0.0001, respectively). Gene set enrichment (GSEA) analysis revealed an enrichment of epithelial to mesenchymal transition and cell cycle progression genes within this cluster, and these patients also had a lower predicted response to immune checkpoint blockade (ICB) (4% vs. 20-34%). An M0-enriched cluster (n = 9) with shorter PFS (p = 0.0006) was also identified in the Clinical Proteomics Tumor Analysis Consortium (CPTAC) cohort (n = 94). Through this characterization of the TIME in ccRCC, a cluster of patients defined by enrichment in M0 macrophages was identified that demonstrated poor prognosis and lower predicted ICB response. Pending further validation, this signature can identify localized ccRCC patients at high risk of recurrence after nephrectomy and who may require therapeutic approaches beyond ICB monotherapy.

Role of adenosine deaminase in prostate cancer progression.

Prostate cancer (PCa) is the second most common cancer and constitutes about 14.7% of total cancer cases. PCa is highly prevalent and more aggressive in African-American (AA) men than in European-American (EA) men. PCa tends to be highly heterogeneous, and its complex biology is not fully understood. We use metabolomics to better understand the mechanisms behind PCa progression and disparities in its clinical outcome. Adenosine deaminase (ADA) is a key enzyme in the purine metabolic pathway; it was found to be upregulated in PCa and is associated with higher-grade PCa and poor disease-free survival. The inosine-to-adenosine ratio, which is a surrogate for ADA activity was high in PCa patient urine and higher in AA PCa compared to EA PCa. To understand the significance of high ADA in PCa, we established ADA overexpression models and performed various in vitro and in vivo studies. Our studies have revealed that an acute increase in ADA expression during later stages of tumor development enhances in vivo growth in multiple pre-clinical models. Further analysis revealed that mTOR signaling activation could be associated with this tumor growth. Chronic ADA overexpression shows alterations in the cells' adhesion machinery and a decrease in cells' ability to adhere to the extracellular matrix in vitro. Losing cell-matrix interaction is critical for metastatic dissemination which suggests that ADA could potentially be involved in promoting metastasis. This is supported by the association of higher ADA expression with higher-grade tumors and poor patient survival. Overall, our findings suggest that increased ADA expression may promote PCa progression, specifically tumor growth and metastatic dissemination.

Molecular Characterization of Clear Cell Renal Cell Carcinoma Reveals Prognostic Significance of Epithelial-mesenchymal Transition Gene Expression Signature.

BACKGROUND: There is an ongoing need to develop prognostic biomarkers to improve the management of clear cell renal cell carcinoma (ccRCC). OBJECTIVE: To leverage enriched pathways in ccRCC to improve risk-stratification. DESIGN, SETTING, AND PARTICIPANTS: We retrospectively identified two complementary discovery cohorts of patients with ccRCC who underwent (1) radical nephrectomy (RNx) with inferior vena cava tumor thrombectomy (patients = 5, samples = 24) and (2) RNx for localized disease and developed recurrence versus no recurrence (n = 36). Patients with localized ccRCC (M0) in The Cancer Genome Atlas (TCGA, n = 386) were used for validation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A differential expression gene (DEG) analysis was performed on targeted RNA next-generation sequencing data from both discovery cohorts. Using TCGA for validation, Kaplan-Meier survival analysis and multivariable Cox proportional hazard testing were utilized to investigate the prognostic impact of DEGs, cell cycle proliferation (CCP), and a novel epithelial-mesenchymal transition (EMT) score on progression-free (PFS) and disease-specific (DSS) survival. RESULTS AND LIMITATIONS: In the discovery cohorts, we observed overexpression of WT1 and CCP genes in the tumor thrombus versus the primary tumor, as well as in patients with recurrence versus those without recurrence. A hallmark pathway analysis demonstrated enrichment of the EMT- and CCP-related pathways in patients with high WT1 expression in the TCGA (validation) ccRCC cohort. CCP and EMT scores were derived in the validation cohort, which was stratified into four risk groups using Youden Index cut points: CCPlow/EMTlow, CCPlow/EMThigh, CCPhigh/EMTlow, and CCPhigh/EMThigh. The CCPhigh/EMThigh risk group was associated with the worst PFS and DSS (both p < 0.001). In a multivariable analysis, CCPhigh/EMThigh was independently associated with poor PFS and DSS (hazard ratio = 4.6 and 10.3, respectively; p < 0.001). CONCLUSIONS: We demonstrate the synergistic prognostic impact of EMT in tumors with a high CCP score. Our novel EMT score has the potential to improve risk stratification and provide potential novel therapeutic targets. PATIENT SUMMARY: Genes involved in epithelial-mesenchymal transition provides important prognostic information for patients with clear cell renal cell carcinoma.

Development of a Whole-urine, Multiplexed, Next-generation RNA-sequencing Assay for Early Detection of Aggressive Prostate Cancer.

BACKGROUND: Despite biomarker development advances, early detection of aggressive prostate cancer (PCa) remains challenging. We previously developed a clinical-grade urine test (Michigan Prostate Score [MiPS]) for individualized aggressive PCa risk prediction. MiPS combines serum prostate-specific antigen (PSA), the TMPRSS2:ERG (T2:ERG) gene fusion, and PCA3 lncRNA in whole urine after digital rectal examination (DRE). OBJECTIVE: To improve on MiPS with a novel next-generation sequencing (NGS) multibiomarker urine assay for early detection of aggressive PCa. DESIGN, SETTING, AND PARTICIPANTS: Preclinical development and validation of a post-DRE urine RNA NGS assay (Urine Prostate Seq [UPSeq]) assessing 84 PCa transcriptomic biomarkers, including T2:ERG, PCA3, additional PCa fusions/isoforms, mRNAs, lncRNAs, and expressed mutations. Our UPSeq model was trained on 73 patients and validated on a held-out set of 36 patients representing the spectrum of disease (benign to grade group [GG] 5 PCa). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The area under the receiver operating characteristic curve (AUC) of UPSeq was compared with PSA, MiPS, and other existing models/biomarkers for predicting GG ≥3 PCa. RESULTS AND LIMITATIONS: UPSeq demonstrated high analytical accuracy and concordance with MiPS, and was able to detect expressed germline HOXB13 and somatic SPOP mutations. In an extreme design cohort (n = 109; benign/GG 1 vs GG ≥3 PCa, stratified to exclude GG 2 cancer in order to capture signal difference between extreme ends of disease), UPSeq showed differential expression for T2:ERG.T1E4 (1.2 vs 78.8 median normalized reads, p < 0.00001) and PCA3 (1024 vs 2521, p = 0.02), additional T2:ERG splice isoforms, and other candidate biomarkers. Using machine learning, we developed a 15-transcript model on the training set (n = 73) that outperformed serum PSA and sequencing-derived MiPS in predicting GG ≥3 PCa in the held-out validation set (n = 36; AUC 0.82 vs 0.69 and 0.69, respectively). CONCLUSIONS: These results support the potential utility of our novel urine-based RNA NGS assay to supplement PSA for improved early detection of aggressive PCa. PATIENT SUMMARY: We have developed a new urine-based test for the detection of aggressive prostate cancer, which promises improvement upon current biomarker tests.

The prevalence and density of asymptomatic Plasmodium falciparum infections among children and adults in three communities of western Kenya.

BACKGROUND: Further reductions in malaria incidence as more countries approach malaria elimination require the identification and treatment of asymptomatic individuals who carry mosquito-infective Plasmodium gametocytes that are responsible for furthering malaria transmission. Assessing the relationship between total parasitaemia and gametocytaemia in field surveys can provide insight as to whether detection of low-density, asymptomatic Plasmodium falciparum infections with sensitive molecular methods can adequately detect the majority of infected individuals who are potentially capable of onward transmission. METHODS: In a cross-sectional survey of 1354 healthy children and adults in three communities in western Kenya across a gradient of malaria transmission (Ajigo, Webuye, and Kapsisywa-Kipsamoite), asymptomatic P. falciparum infections were screened by rapid diagnostic tests, blood smear, and quantitative PCR of dried blood spots targeting the varATS gene in genomic DNA. A multiplex quantitative reverse-transcriptase PCR assay targeting female and male gametocyte genes (pfs25, pfs230p), a gene with a transcriptional pattern restricted to asexual blood stages (piesp2), and human GAPDH was also developed to determine total parasite and gametocyte densities among parasitaemic individuals. RESULTS: The prevalence of varATS-detectable asymptomatic infections was greatest in Ajigo (42%), followed by Webuye (10%). Only two infections were detected in Kapsisywa. No infections were detected in Kipsamoite. Across all communities, children aged 11-15 years account for the greatest proportion total and sub-microscopic asymptomatic infections. In younger age groups, the majority of infections were detectable by microscopy, while 68% of asymptomatically infected adults (> 21 years old) had sub-microscopic parasitaemia. Piesp2-derived parasite densities correlated poorly with microscopy-determined parasite densities in patent infections relative to varATS-based detection. In general, both male and female gametocytaemia increased with increasing varATS-derived total parasitaemia. A substantial proportion (41.7%) of individuals with potential for onward transmission had qPCR-estimated parasite densities below the limit of microscopic detection, but above the detectable limit of varATS qPCR. CONCLUSIONS: This assessment of parasitaemia and gametocytaemia in three communities with different transmission intensities revealed evidence of a substantial sub-patent infectious reservoir among asymptomatic carriers of P. falciparum. Experimental studies are needed to definitively determine whether the low-density infections in communities such as Ajigo and Webuye contribute significantly to malaria transmission.

Tumor size and genomic risk in localized prostate cancer.

PURPOSE: Unlike many other cancers, measurement of primary prostate tumor size has no defined role in the management of localized prostate cancer. Here, we assess whether prostate tumor size is associated with aggressive tumor biology using biomarkers of genomic risk. MATERIALS AND METHODS: We abstracted or imputed tumor size from the primary pathology reports of prostate cancers incorporated in The Cancer Genome Atlas. We used transcriptomic data to estimate the Cell Cycle Progression Score (CCPS, Prolaris), the Genomic Classifier Score (GCS, Decipher) and the Genomic Prostate Score (GPS, OncotypeDx), SChLaP1 expression, and copy number alteration percentage (%CNA) as well as hallmark gene set enrichment analysis. RESULTS: Tumor size and gene expression data was available for 267 men. On multivariable regression adjusted for Gleason Grade Group and tumor purity, tumor size was independently associated with the calculated (c)GCS, cGPS, SChLaP1 expression, and %CNA (P< 0.05), but not cCCPS. Gene set enrichment analysis demonstrated that tumors <5 cc, when adjusting for Gleason grade group, were enriched for androgen response genes, while tumors >5 cc were enriched for MYC targets and genes associated with epithelial mesenchymal transition. CONCLUSIONS: Prostate tumor size is independently associated with established markers of genomic risk. This study nominates the size of a primary prostate cancer as candidate for inclusion in future novel risk scores seeking to quantify cancer aggressiveness.

Multigene Profiling of Circulating Tumor Cells (CTCs) for Prognostic Assessment in Treatment-Naïve Metastatic Hormone-Sensitive Prostate Cancer (mHSPC).

The substantial biological heterogeneity of metastatic prostate cancer has hindered the development of personalized therapeutic approaches. Therefore, it is difficult to predict the course of metastatic hormone-sensitive prostate cancer (mHSPC), with some men remaining on first-line androgen deprivation therapy (ADT) for several years while others progress more rapidly. Improving our ability to risk-stratify patients would allow for the optimization of systemic therapies and support the development of stratified prospective clinical trials focused on patients likely to have the greatest potential benefit. Here, we applied a liquid biopsy approach to identify clinically relevant, blood-based prognostic biomarkers in patients with mHSPC. Gene expression indicating the presence of CTCs was greater in CHAARTED high-volume (HV) patients (52% CTChigh) than in low-volume (LV) patients (23% CTChigh; * p = 0.03). HV disease (p = 0.005, q = 0.033) and CTC presence at baseline prior to treatment initiation (p = 0.008, q = 0.033) were found to be independently associated with the risk of nonresponse at 7 months. The pooled gene expression from CTCs of pre-ADT samples found AR, DSG2, KLK3, MDK, and PCA3 as genes predictive of nonresponse. These observations support the utility of liquid biomarker approaches to identify patients with poor initial response. This approach could facilitate more precise treatment intensification in the highest risk patients.

Serial Molecular Profiling of Low-grade Prostate Cancer to Assess Tumor Upgrading: A Longitudinal Cohort Study.

BACKGROUND: The potential for low-grade (grade group 1 [GG1]) prostate cancer (PCa) to progress to high-grade disease remains unclear. OBJECTIVE: To interrogate the molecular and biological features of low-grade PCa serially over time. DESIGN, SETTING, AND PARTICIPANTS: Nested longitudinal cohort study in an academic active surveillance (AS) program. Men were on AS for GG1 PCa from 2012 to 2017. INTERVENTION: Electronic tracking and resampling of PCa using magnetic resonance imaging/ultrasound fusion biopsy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: ERG immunohistochemistry (IHC) and targeted DNA/RNA next-generation sequencing were performed on initial and repeat biopsies. Tumor clonality was assessed. Molecular data were compared between men who upgraded and those who did not upgrade to GG ≥ 2 cancer. RESULTS AND LIMITATIONS: Sixty-six men with median age 64 yr (interquartile range [IQR], 59-69) and prostate-specific antigen 4.9 ng/mL (IQR, 3.3-6.4) underwent repeat sampling of a tracked tumor focus (median interval, 11 mo; IQR, 6-13). IHC-based ERG fusion status was concordant at initial and repeat biopsies in 63 men (95% vs expected 50%, p < 0.001), and RNAseq-based fusion and isoform expression were concordant in nine of 13 (69%) ERG+ patients, supporting focal resampling. Among 15 men who upgraded with complete data at both time points, integrated DNA/RNAseq analysis provided evidence of shared clonality in at least five cases. Such cases could reflect initial undersampling, but also support the possibility of clonal temporal progression of low-grade cancer. Our assessment was limited by sample size and use of targeted sequencing. CONCLUSIONS: Repeat molecular assessment of low-grade tumors suggests that clonal progression could be one mechanism of upgrading. These data underscore the importance of serial tumor assessment in men pursuing AS of low-grade PCa. PATIENT SUMMARY: We performed targeted rebiopsy and molecular testing of low-grade tumors on active surveillance. Our findings highlight the importance of periodic biopsy as a component of monitoring for cancer upgrading during surveillance.

Whole-Blood Transcriptional Signatures Composed of Erythropoietic and NRF2-Regulated Genes Differ Between Cerebral Malaria and Severe Malarial Anemia.

Background: Among the severe malaria syndromes, severe malarial anemia (SMA) is the most common, whereas cerebral malaria (CM) is the most lethal. However, the mechanisms that lead to CM and SMA are unclear. Methods: We compared transcriptomic profiles of whole blood obtained from Ugandan children with acute CM (n = 17) or SMA (n = 17) and community children without Plasmodium falciparum infection (n = 12) and determined the relationships among gene expression, hematological indices, and relevant plasma biomarkers. Results: Both CM and SMA demonstrated predominantly upregulated enrichment of dendritic cell activation, inflammatory/Toll-like receptor/chemokines, and monocyte modules, but downregulated enrichment of lymphocyte modules. Nuclear factor, erythroid 2 like 2 (Nrf2)-regulated genes were overexpressed in children with SMA relative to CM, with the highest expression in children with both SMA and sickle cell disease (HbSS), corresponding with elevated plasma heme oxygenase-1 in this group. Erythroid and reticulocyte-specific signatures were markedly decreased in CM relative to SMA despite higher hemoglobin levels and appropriate increases in erythropoietin. Viral sensing/interferon-regulatory factor 2 module expression and plasma interferon-inducible protein-10/CXCL10 negatively correlated with reticulocyte-specific signatures. Conclusions: Compared with SMA, CM is associated with downregulation of Nrf2-related and erythropoiesis signatures by whole-blood transcriptomics. Future studies are needed to confirm these findings and assess pathways that may be amenable to interventions to ameliorate CM and SMA.

Biologic Significance of Magnetic Resonance Imaging Invisibility in Localized Prostate Cancer.

PURPOSE: Multiparametric magnetic resonance imaging (mpMRI) is used widely for prostate cancer (PCa) evaluation. Approximately 35% of aggressive tumors, however, are not visible on mpMRI. We sought to identify the molecular alterations associated with mpMRI-invisible tumors and determine whether mpMRI visibility is associated with PCa prognosis. METHODS: Discovery and validation cohorts included patients who underwent mpMRI before radical prostatectomy and were found to harbor both mpMRI-visible (Prostate Imaging and Reporting Data System 3 to 5) and -invisible (Prostate Imaging and Reporting Data System 1 or 2) foci on surgical pathology. Next-generation sequencing was performed to determine differential gene expression between mpMRI-visible and -invisible foci. A genetic signature for tumor mpMRI visibility was derived in the discovery cohort and assessed in an independent validation cohort. Its association with long-term oncologic outcomes was evaluated in a separate testing cohort. RESULTS: The discovery cohort included 10 patients with 26 distinct PCa foci on surgical pathology, of which 12 (46%) were visible and 14 (54%) were invisible on preoperative mpMRI. Next-generation sequencing detected prioritized genetic mutations in 14 (54%) tumor foci (n = 8 mpMRI visible, n = 6 mpMRI invisible). A nine-gene signature (composed largely of cell organization/structure genes) associated with mpMRI visibility was derived (area under the curve = 0.89), and the signature predicted MRI visibility with 75% sensitivity and 100% specificity (area under the curve = 0.88) in the validation cohort. In the testing cohort (n = 375, median follow-up 8 years) there was no significant difference in biochemical recurrence, distant metastasis, or cancer-specific mortality in patients with predicted mpMRI-visible versus -invisible tumors (all P > .05). CONCLUSION: Compared with mpMRI-invisible disease, mpMRI-visible tumors are associated with underexpression of cellular organization genes. mpMRI visibility does not seem to be predictive of long-term cancer outcomes, highlighting the need for biopsy strategies that detect mpMRI-invisible tumors.

Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a, Il1b, and Tnfa, the chemokine genes Cxcl2 and Ccl5, and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.