Analytical Validation of SOD2 Genotyping.

Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells' protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.

Val16A SOD2 Polymorphism Promotes Epithelial-Mesenchymal Transition Antagonized by Muscadine Grape Skin Extract in Prostate Cancer Cells.

Epithelial-mesenchymal transition (EMT), a key event in cancer metastasis, allows polarized epithelial cells to assume mesenchymal morphologies, enhancing invasiveness and migration, and can be induced by reactive oxygen species (ROS). Val16A (Ala) SOD2 polymorphism has been associated with increased prostate cancer (PCa) risk. We hypothesized that SOD2 Ala single nucleotide polymorphism (SNP) may promote EMT. We analyzed SOD2 expression and genotype in various prostate cell lines. Stable overexpression of Ala-SOD2 or Val-SOD2 allele was performed in Lymph Node Carcinoma of the Prostate (LNCaP) cells followed by analysis of intracellular ROS and EMT marker protein expression. Treatments were performed with muscadine grape skin extract (MSKE) antioxidant, with or without addition of H2O2 to provide further oxidative stress. Furthermore, MTS cell proliferation, cell migration, and apoptosis assays were completed. The results showed that SOD2 expression did not correlate with tumor aggressiveness nor SOD2 genotype. We demonstrated that the Ala-SOD2 allele was associated with marked induction of EMT indicated by higher Snail and vimentin, lower E-cadherin, and increased cell migration, when compared to Val-SOD2 allele or Neo control cells. Ala-SOD2 SNP cells exhibited increased levels of total ROS and superoxide and were more sensitive to co-treatment with H2O2 and MSKE, which led to reduced cell growth and increased apoptosis. Additionally, MSKE inhibited Ala-SOD2 SNP-mediated EMT. Our data indicates that treatment with a combination of H2O2-generative drugs, such as certain chemotherapeutics and antioxidants such as MSKE that targets superoxide, hold promising therapeutic potential to halt PCa progression in the future.

A pilot study of prostate-specific membrane antigen (PSMA) dynamics in men undergoing treatment for advanced prostate cancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a rational target for noninvasive detection of recurrent prostate cancer (PCa) and for therapy of metastatic castration-resistant prostate cancer (mCRPC) with PSMA-targeted agents. Here we conducted serial measurements of PSMA expression on circulating tumor cells (CTCs) to evaluate patterns of longitudinal PSMA dynamics over the course of multiple sequential therapies. METHODS: A retrospective investigation of men with mCRPC undergoing evaluation at medical oncology clinics at our institution assessed the dynamics of PSMA expression in the context of different systemic treatments administered sequentially. Eligibility included patients who began systemic therapies with androgen receptor (AR)-directed agents or taxane agents for whom peripheral blood samples were tested for CTC mRNA of AR splice variant-7 (AR-V7), prostate-specific antigen (PSA), and PSMA (with >2 CTC + results) in a CLIA-accredited laboratory. RESULTS: From August 2015 to November 2017, we identified 96 eligible men. Fifteen had greater than or equal to 2 sequential therapies and evaluable CTC samples, greater than or equal to 1 expressing PSMA (PSMA+). Among the 15 patients included in this analysis, a total of 54 PSMA status evaluations were performed in the context of 48 therapies during a median follow-up of 18 months. At baseline, PSMA signal was detected ("positive") in 11 of 15 (73.3%) patients, while for 4 of 15 (26.7%) patients PSMA signal was undetectable ("negative"). In all but two patients, the baseline collection corresponded with a change in treatment. On the second assessment, PSMA increases were detected in all 4/4 (100%) PSMA-negative patients and 8 of 11 (72.7%) PSMA-positive patients. PSMA significantly decreased in a patient treated with 177 Lu-PSMA-617. Serum PSA declines were seen in 7 of 8 (88%) of the treatment periods where PSMA decreased. CONCLUSIONS: PSMA expression in CTCs is a dynamic marker. PSMA transcript declines appear to be associated with concurrent decreases in serum PSA. Sequential CTC sampling could provide a noninvasive response assessment to systemic treatment for mCRPC.

Validation Strategy for Ultrasensitive Mutation Detection.

BACKGROUND: Ultrasensitive detection of low-abundance DNA point mutations is a challenging molecular biology problem, because nearly identical mutant and wild-type molecules exhibit crosstalk. Reliable ultrasensitive point mutation detection will facilitate early detection of cancer and therapeutic monitoring of cancer patients. OBJECTIVE: The objective of this study was to develop a method to correct errors in low-level cell line mixes. MATERIALS AND METHODS: We tested sample mixes with digital-droplet PCR (ddPCR) and next-generation sequencing. RESULTS: We introduced two corrections: baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation; and haplotype counting was used to correct errors in cell counting and pipetting. We found ddPCR to have better correlation for detecting low-level mutations without applying any correction (R2 = 0.80) and be more linear after introducing both corrections (R2 = 0.99). CONCLUSIONS: The VAF correction was found to be more significant than haplotype correction. It is imperative that various technologies be evaluated against each other and laboratories be provided with defined quality control samples for proficiency testing.

Differential preventive activity of sulindac and atorvastatin in Apc+/Min-FCCCmice with or without colorectal adenomas.

OBJECTIVE: The response of subjects to preventive intervention is heterogeneous. The goal of this study was to determine if the efficacy of a chemopreventive agent differs in non-tumour-bearing animals versus those with colorectal tumours. Sulindac and/or atorvastatin was administered to Apc+/Min-FCCC mice with known tumour-bearing status at treatment initiation. DESIGN: Male mice (6-8 weeks old) underwent colonoscopy and received control chow or chow with sulindac (300 ppm), atorvastatin (100 ppm) or sulindac/atorvastatin. Tissues were collected from mice treated for 14 weeks (histopathology) or 7 days (gene expression). Cell cycle analyses were performed on SW480 colon carcinoma cells treated with sulindac, atorvastatin or both. RESULTS: The multiplicity of colorectal adenomas in untreated mice bearing tumours at baseline was 3.6-fold higher than that of mice that were tumour free at baseline (P=0.002). Atorvastatin completely inhibited the formation of microadenomas in mice that were tumour free at baseline (P=0.018) and altered the expression of genes associated with stem/progenitor cells. Treatment of tumour-bearing mice with sulindac/atorvastatin led to a 43% reduction in the multiplicity of colorectal adenomas versus untreated tumour-bearing mice (P=0.049). Sulindac/atorvastatin increased the expression of Hoxb13 and Rprm significantly, suggesting the importance of cell cycle regulation in tumour inhibition. Treatment of SW480 cells with sulindac/atorvastatin led to cell cycle arrest (G0/G1). CONCLUSIONS: The tumour status of animals at treatment initiation dictates response to therapeutic intervention. Atorvastatin eliminated microadenomas in tumour-free mice. The tumour inhibition observed with Sul/Atorva in tumour-bearing mice was greater than that achieved with each agent.

Clinical mutational profiling of 1006 lung cancers by next generation sequencing.

Analysis of lung adenocarcinomas for actionable mutations has become standard of care. Here, we report our experience using next generation sequencing (NGS) to examine AKT1, BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA genes in 1006 non-small cell lung cancers in a clinical diagnostic setting. NGS demonstrated high sensitivity. Among 760 mutations detected, the variant allele frequency (VAF) was 2-5% in 33 (4.3%) mutations and 2-10% in 101 (13%) mutations. A single bioinformatics pipeline using Torrent Variant Caller, however, missed a variety of EGFR mutations. Mutations were detected in KRAS (36% of tumors), EGFR (19%) including 8 (0.8%) within the extracellular domain (4 at codons 108 and 4 at codon 289), BRAF (6.3%), and PIK3CA (3.7%). With a broader reportable range, exon 19 deletion and p.L858R accounted for only 36% and 26% of EGFR mutations and p.V600E accounted for only 24% of BRAF mutations. NGS provided accurate sequencing of complex mutations seen in 19% of EGFR exon 19 deletion mutations. Doublet (compound) EGFR mutations were observed in 29 (16%) of 187 EGFR-mutated tumors, including 69% with two non-p.L858R missense mutations and 24% with p.L858 and non-p.L858R missense mutations. Concordant VAFs suggests doublet EGFR mutations were present in a dominant clone and cooperated in oncogenesis. Mutants with predicted impaired kinase, observed in 25% of BRAF-mutated tumors, were associated with a higher incidence of concomitant activating KRAS mutations. NGS demonstrates high analytic sensitivity, broad reportable range, quantitative VAF measurement, single molecule sequencing to resolve complex deletion mutations, and simultaneous detection of concomitant mutations.

Analytical Validation of Androgen Receptor Splice Variant 7 Detection in a Clinical Laboratory Improvement Amendments (CLIA) Laboratory Setting.

Patients with castration-resistant prostate cancer (CRPC) often are treated with drugs that target the androgen receptor (AR) ligand-binding domain. Constitutively active AR splice variant 7 (AR-V7) lacks the ligand-binding domain and, if detected in circulating tumor cells, may be associated with resistance to these agents. We validated an AR-V7 assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Circulating tumor cells were isolated, and mRNA was reverse-transcribed into cDNA. Real-time quantitative PCR amplification of reference transcripts (beta-actin and glyceraldehyde-3-phosphate dehydrogenase), prostate-specific transcripts (prostate-specific membrane antigen, prostate-specific antigen, and AR-full length), and AR-V7 was performed. Specimens for validation included an AR-V7 expressing prostate cancer (LNCaP95), 38 peripheral blood controls, and 21 blood samples from CRPC patients. The assay detected as few as five LNCaP95 cells spiked into peripheral blood, showing high analytical sensitivity. Multiple inter-run and intrarun replicates of LNCaP95 cell line experiments yielded similar cycle threshold values for all genes, showing high analytical precision (AR-V7 cycle threshold CV of 0.67%). All 38 healthy control samples were negative for AR-V7, showing high diagnostic specificity (100%). The diagnostic accuracy was confirmed by concurrent testing of 21 CRPC samples in the research laboratory and the clinical diagnostic laboratory: concordance in AR-V7 status was achieved in all cases (positive in 4, negative in 17) (100% accuracy). This first validated clinical assay detects the AR-V7 with high analytical sensitivity, precision, specificity, and accuracy.

Estrogen metabolism in the human lung: impact of tumorigenesis, smoke, sex and race/ethnicity.

Previous data from this group demonstrate that the murine lung metabolizes estrogen. Production of the putative carcinogen 4-hydroxyestrogen (4-OHE) is elevated within the lungs of female vs. male mice and accelerated by tobacco smoke. The goal of this study was to determine if the human lung metabolizes estrogen and evaluate the impact of tumor formation, smoke, sex and race/ethnicity on metabolism. Urine and lung tissue (normal, tumor) were obtained from 49 non-small cell lung cancer patients. Healthy postmenopausal Caucasian (n = 19) and Chinese (n = 20) American women (never-smokers) donated urine. Quantitative RT-PCR analyses indicate that multiple estrogen synthesis and metabolism genes are expressed in human bronchoalveolar cells. Estrogen and its metabolites were measured in lung tissue and urine using liquid chromatography/tandem mass spectrometry. Wilcoxon rank tests were used for statistical comparisons. E1, E2, E3 and estrogen metabolites 2-OHE1, 2-OHE2, 4-OHE1, 4-OHE2, 2-OME1 and 2-OME2 were detected at higher levels in tumor vs. adjacent normal tissue and in women vs. men (P < 0.05). The proportion of 4-OHEs was higher in tumors than in normal lung tissue (P < 0.05), and elevated in normal tissue from current- vs. never-smoking women (P = 0.006); similar trends were observed in urine. The proportion of 4-OHEs in the urine of postmenopausal Chinese American women was 1.8-fold higher than that of Caucasian women (P = 0.015). These data indicate that estrogen metabolites are present in the human lung. A shift towards 4-hydroxylation during lung tumorigenesis may contribute to the risk conferred by smoking, sex or race/ethnicity.