TaqMan Copy Number Assay Research Articles

Pharmacogenetics and pharmacokinetics of tamoxifen in a Zimbabwean breast cancer cohort.

Tamoxifen is the most used hormonal therapy for oestrogen receptor-positive breast cancer. CYP2D6 is the main enzyme in the metabolic pathway of tamoxifen to endoxifen. Variations in endoxifen plasma concentrations are associated with CYP2D6 polymorphisms. This study aimed to determine the association between the CYP2D6 polymorphisms and endoxifen plasma concentrations in a cohort of Zimbabwean breast cancer patients (n= 40). TaqMan genotyping and copy number assays were done to determine CYP2D6 genotypes. Tamoxifen and metabolites were quantitated using LC-MS/MS. The population had high frequencies of the CYP2D6 reduced function alleles, *17 (15%) and *29 (18%). The median endoxifen concentration was 4.78 ng/mL, and in 55% of the patients, mostly intermediate metabolizers were below the endoxifen therapeutic threshold of 5.97 ng/mL. The CYP2D6 phenotypes and activity scores were significantly associated with endoxifen plasma concentrations (P= 0.0151) and with endoxifen to N-desmethyl-tamoxifen ratios (P= 0.0006).

Genotyping, characterization, and imputation of known and novel CYP2A6 structural variants using SNP array data.

CYP2A6 metabolically inactivates nicotine. Faster CYP2A6 activity is associated with heavier smoking and higher lung cancer risk. The CYP2A6 gene is polymorphic, including functional structural variants (SV) such as gene deletions (CYP2A6*4), duplications (CYP2A6*1 × 2), and hybrids with the CYP2A7 pseudogene (CYP2A6*12, CYP2A6*34). SVs are challenging to genotype due to their complex genetic architecture. Our aims were to develop a reliable protocol for SV genotyping, functionally phenotype known and novel SVs, and investigate the feasibility of CYP2A6 SV imputation from SNP array data in two ancestry populations. European- (EUR; n = 935) and African- (AFR; n = 964) ancestry individuals from smoking cessation trials were genotyped for SNPs using an Illumina array and for CYP2A6 SVs using Taqman copy number (CN) assays. SV-specific PCR amplification and Sanger sequencing was used to characterize a novel SV. Individuals with SVs were phenotyped using the nicotine metabolite ratio, a biomarker of CYP2A6 activity. SV diplotype and SNP array data were integrated and phased to generate ancestry-specific SV reference panels. Leave-one-out cross-validation was used to investigate the feasibility of CYP2A6 SV imputation. A minimal protocol requiring three Taqman CN assays for CYP2A6 SV genotyping was developed and known SV associations with activity were replicated. The first domain swap CYP2A6-CYP2A7 hybrid SV, CYP2A6*53, was identified, sequenced, and associated with lower CYP2A6 activity. In both EURs and AFRs, most SV alleles were identified using imputation (>70% and >60%, respectively); importantly, false positive rates were <1%. These results confirm that CYP2A6 SV imputation can identify most SV alleles, including a novel SV.

Utility of real-time polymerase chain reaction for the assessment of CDKN2A homozygous deletion in adult-type IDH-mutant astrocytoma.

The World Health Organization Classification of Tumors of the Central Nervous System 5th Edition (WHO CNS5) introduced a newly defined astrocytoma, IDH-mutant grade 4, for adult diffuse glioma classification. One of the diagnostic criteria is the presence of a CDKN2A/B homozygous deletion (HD). Here, we report a robust and cost-effective quantitative polymerase chain reaction (qPCR)-based test for assessing CDKN2A HD. A TaqMan copy number assay was performed using a probe located within CDKN2A. The linear correlation between the Ct values and relative CDKN2A copy number was confirmed using a serial mixture of DNA from normal blood and U87MG cells. The qPCR assay was performed in 109 IDH-mutant astrocytomas, including 14 tumors with CDKN2A HD, verified either by multiplex ligation-dependent probe amplification (MLPA) or CytoScan HD microarray platforms. Receiver operating characteristic curve analysis indicated that a cutoff value of 0.85 yielded optimal sensitivity (100%) and specificity (99.0%) for determining CDKN2A HD. The assay applies to DNA extracted from frozen or formalin-fixed paraffin-embedded tissue samples. Survival was significantly shorter in patients with than in those without CDKN2A HD, assessed by either MLPA/CytoScan or qPCR. Thus, our qPCR method is clinically applicable for astrocytoma grading and prognostication, compatible with the WHO CNS5.

CSIG-10. PLATELET-DERIVED GROWTH FACTOR RECEPTOR ALPHA ONCOGENE DEPENDENCY IN GLIOBLASTOMA

Abstract PDGFRA is the second most frequently amplified gene encoding receptor tyrosine kinase in adult glioblastoma (GBM), oftentimes as extrachromosomal elements (ecDNA). Our overall objective is to elucidate mechanisms underlying PDGFRα dependency in GBM tumor maintenance. We have isolated distinct subpopulations from a GBM model (HF3253), harboring two alterations in PDGFRA: constitutively active genomic rearrangement and extrachromosomal amplification, that differ in the frequency of PDGFRA ecDNA. HF3253 tumor growth rate correlates with the initial proportion of ecDNA+ population implanted. Furthermore, slower tumor growth is due to selection for initially low-frequency PDGFRA ecDNA amplified clones based on histology and TaqMan Copy Number assay. Further exploiting intra-tumoral heterogeneity, we have isolated single cell clones from bulk cells. Compared to bulk cells, single cell clones do not express PDGFRα, PDGFRA mRNA and exhibit diploid PDGFRA copy number. Tumor growth was reduced in 4 ecDNA(-) clones compared to parental ecDNA(+) (log-rank test p= 0.00772, 0.00379, 0.00076, 0.00379). In contrast to parental HF3253, ecDNA(-) tumors demonstrated diffuse tumor morphology and weak PDGFRα activation. HF3253 ecDNA(-) PDX tumors lack detectable PDGFRα. Correspondingly, HF3253 ecDNA(-) cell populations do not exhibit de novo PDGFRA copy number gains post-implant. We conducted paired, whole RNA-sequencing on 20 HF3253 populations (ecDNA+/-: 6 clones from 3 biological replicates PDXs and 4 clones from 4 in vitro technical replicates). Employing a false discovery rate of 0.05, we identified 785 differentially expressed genes. Platelet-derived growth factor binding (GO:0048407) and central carbon metabolism were down-regulated in ecDNA(-) while genes significantly associated with astrocytic processes were upregulated. We demonstrated the dependency on PDGFRα signaling in a patient-derived GBM model carrying ecDNA PDGFRA amplification. Our data validates PDGFRɑ as a therapeutic target in a subset of GBM patients and demonstrates that detection of ecDNA-amplified PDGFRA has the potential to be a predictive biomarker of future PDGFRɑ targeted therapies.

Abstract 2504: Functional characterization of chromosomal deletions in head and neck squamous cell carcinoma

Abstract Introduction: Large-scale chromosomal deletions frequently occur in the progression of head and neck squamous cell carcinoma (HNSCC). Many studies have linked these deletions with particular patient outcomes. However, their precise contributions to tumorigenesis have yet to be elucidated. Here, we introduce an in vitro model for studying large-scale chromosomal deletions to establish their role as driver or passenger events in HNSCC progression. Methods: CRISPR/Cas9 vectors coding for guide RNAs (gRNAs) that flank two of the commonly deleted regions in HNSCC tumors were transfected into primary human keratinocytes. Eighteen different combinations of gRNAs were utilized to create deletions of various sizes at two different genomic loci. Genomic DNA (gDNA) was harvested 24 hours post-transfection and analyzed via endpoint PCR and Sanger sequencing to assess for the presence of the deletions. Edited cells were further sorted via fluorescence-activated cell sorting (FACS) and the efficiency of the deletions were analyzed via performing TaqMan copy number assay qPCR on extracted gDNA. Subsequently, the invasive properties of the genome-edited cells were then investigated using organotypic cultures, a near-physiological three-dimensional model system for studying squamous epithelial biology. Results: The feasibility of performing large-scale deletions was confirmed via endpoint PCR and Sanger sequencing, in which all 18 gRNA combinations created successful deletions. We further sought to create a pure population of the cells harboring the deletions by isolating the edited cells via FACS. TaqMan copy number assay qPCR on gDNA harvested from these cells indicated a 10-45% reduction in the copy number of loci of interest. Genome-edited keratinocytes containing deletions at two of the most commonly deleted sites in HNSCC tumors were used for organotypic assays, in which the preliminary results suggested that deletion of these sites can lead to a gain of invasive behavior. Conclusion: Chromosomal deletions occur at a high frequency in HNSCC tumors. However, there is no established model to determine whether these deletions exist as driver or passenger events. Here, we demonstrate the introduction of successful large-scale deletions in a primary human cell culture system that can be further utilized to study the role of these events in HNSCC tumorigenesis. Furthermore, we show that two of the most common deletions in HNSCC may act as driver events in tumor progression. Support: This study was supported by Program in Breakthrough Biological Research, UCSF School of Dentistry OHNRG pilot grant, and an R01 grant (DE029890-01 - Defining the Mutational pathogenesis of Oral Preneoplasia). Citation Format: Ramin Farhad, Vicente Planells Palop, Aaron Tward. Functional characterization of chromosomal deletions in head and neck squamous cell carcinoma [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 2504.

Microdeletion in Chromosome 17 Cytoband q21.31 Identified As Located within the KANSL1 Gene Prevailed in Flt3-ITD Negative Normal Karyotype AML Patients As Opposed to Their AML Counterparts and Influenced the Phenotype of the Disease

Recent advances in genetic tools for leukemic diagnosis have facilitated studies on genetic traits which in concert with a driving mutation if present may influence the phenotype of the disease. Flt3-ITD is present in about 30% of AML patients and tends to be associated with a poor prognosis. Currently the use of inhibitors of mutated and in consequence activated kinases is undergoing clinical trials. The dim prognosis of Flt3-ITD positive AML and the hope associated with kinases inhibitors prompted us to evaluate in some depth the genetics of AML cases in relation to the presence of Flt3-ITD .Ninety-two AML patients (diagnosed according to the FAB/WHO criteria, F/M=49/43, age median: 57, range: 21-81 years) who were seen in our institution were recruited to the present study which was conducted as follows:The patients underwent GTG karyotyping and/or FISH for X or Y deletion, inv (3), -5/5q-, -7/7q-,+8, MLL, RUNX1, PML/RARA or RARA, inv(16). 52 lacking GTG and/or FISH abnormalities were called as normal karyotype (NK) patients, among those, 20 were Flt3-ITD positive. The other 40 patients had karyotype abnormalities or were FISH positive for AML-associated abnormalities among which 10 had Flt3-ITD .In all 92 patients microarray analysis was performed using Agilent - Catalog Agilent Cancer CGH+SNP 180K or Roche - WG Catalog NimbleGene 12x270K microarrays.We found (Partec Genomics Suite and Biodiscovery Nexus 8 software) that NK Flt3-ITD negative had, when the whole genome was analyzed, a higher number of total copy number variation (CNV) aberrations (amplifications and/or deletions, vs 32.21±8.70 vs 16.15±2.60, p=0.008) including one copy losses, (2.79±0.57 vs 1.20±0.35 p=0.019)as opposed to NK patients having Flt3-ITD mutation .During the study, an interim analysis was performed at the time we switched from Roche to Agilent microarrays which revealed the significant prevalence of CNVs in AML Flt3-ITD negative patients over Flt3-ITD positive ones within the region 17q21.31 specifically in the positions from 44281092 to 4476549, 44702953 to 44745272 and 44745272 to 44765496. The same region appeared again as significantly discriminating Flt3-ITD negative and positive patients at the final analysis. Therefore, this region was analyzed in some depth. Hg19 annotation was used and according to that the region encompasses the KANSL1 gene among two others (NSFP1 and NSF). The NSF gene encodes a protein involved in transport to the Golgi and NSFP1 is a pseudogene. KANSL1, a component of the MLL1 complex (thought to contribute to leukemogenesis), was the focus of our interest, and the gene was further investigated to validate the microarray-based platform results and to localize the CNV area more precisely. In the region 44281092 to 4476549 within the KANSL1 gene Flt3-ITD negative patients had more deletions and amplifications as compared to those Flt3-ITD positive (31/62 vs 7/30, p=0.012). The same was valid when only deletions were considered (19/62 vs 4/30, p=0.058).Then to validate microarray platform results the same 80 samples were examined using the TaqMan Copy Number Assay (ThermoFisher) for the KANSL1 gene (primers and probe designed for the region 44297111-44297201 lying in chr17), and as a reference the RNaseP gene was employed. The results obtained using the microarray platform correlated well (r=0.671, p<0.001) with those obtained using RT-PCR copy number assay for KANSL1 .61 DNA samples obtained from the patients' solid organ biopsies were examined using the Copy Number Assay for KANSL1 gene CNV. In all instances except one the same CNP was observed in the marrow as that in the solid organ biopsies. It lends credit to the technology employed in this study, but still we do not have enough data to exclude the somatic origin of the microdeletions.The microdeletions are of biologic significance, as suggested by the following findings: 1.All three patients with FISH-documented MLL amplification had deletions within the KANSL1 gene (chr:17 44281092 to 4476549).2.Microdeletions within the KANSL1 gene were more frequently observed in the group M0-M2 patients than in other subtypes of AML (18/51 vs 5/35, p=0.026).In conclusion, a lack of Flt3-ITD and normal karyotype was associated with the frequent presence of microdeletions within the chr17 cytoband q21.31. The latter was identified as (i) present within the KANSL1 gene and (ii) influencing the phenotype of AML.Supported by the INNOMED/I/1/NCBR/2014 DisclosuresNo relevant conflicts of interest to declare.

TP53 mutations Are Associated with Mutated MYD88 and CXCR4, and Confer an Adverse Outcome in Waldenström Macroglobulinemia

Background: Whole genome sequencing identified highly recurrent somatic mutations in Waldenström macroglobulinemia (WM). Activating somatic mutations in MYD88 and CXCR4 are present in 90-95% and 30-40% of WM patients, respectively, and impact disease presentation, treatment outcome, and overall survival. In contrast, somatic mutations in the tumor suppressor gene TP53 occur less frequently (7%), and the clinical significance remains to be delineated. TP53 aberrations confer an adverse prognosis in related B-cell malignancies, such as chronic lymphocytic leukemia and multiple myeloma, predicting for treatment resistant and inferior survival. As such, we sought to characterize the clinical implications, as well as the clonal architecture, of somatic TP53 mutations in WM. Methods: We searched our database for WM patients with a TP53 mutation identified by a clinical next generation sequencing (NGS) assay using unsorted bone marrow (BM) samples. To validate the findings, CD19+ cells from BM aspirates were isolated, and DNA was extracted for mutational analysis. CD19-depleted peripheral blood (PB) mononuclear cells were used as normal paired samples. All samples were screened for MYD88, CXCR4, and TP53 mutations by Sanger sequencing, and zygosity was determined by establishing the ratio of mutant versus wile-type (WT) allele expression. TP53 copy number was determined using TaqMan Copy Number Assays (Applied Biosystems, Grand Island, NY, USA). Medical files were manually reviewed to gather pertinent clinical data. Results: Fourteen WM patients (14/265; 5.3%) had a TP53 mutation detected by a clinical NGS assay. Sanger sequencing identified somatic TP53 mutations within the WM clone in six patients, including one patient with two somatic mutations. Three patients had a TP53 mutation identified in both CD19+ and CD19- sorted samples, 4 patients were WT, and 1 patient did not have DNA for validation. No recurrent variants were identified. Among the six patients with validated somatic TP53 mutations, four (67%) had biallelic inactivation of TP53 . Three patients had a homozygous TP53 mutation determined by Sanger sequencing and one patient had deletion of TP53 . All six patients harbored both a CXCR4 mutation (4 nonsense, 2 frameshift) and the MYD88 L265P mutation, of which 4 patients (67%) had mutated homozygous MYD88 . At the time the TP53 mutation was detected, the median BM involvement and hemoglobin level was 80% and 9.2 g/dl, respectively. The median serum IgM level was 2,508 mg/dl, and two patients had symptomatic hyperviscosity. Five patients (83%) were high-risk according to the International Prognostic Scoring System for WM. Three patients were untreated, 2 patients were refractory to their most recent therapy (i.e. bortezomib, dexamethasone, rituximab), and 1 patient was relapsing. Four patients went on to receive systemic therapy, to which two patients were refractory. Two patients were treated with ibrutinib and both obtained a major response. After a median follow-up 7.7 months, 2 patients (33%) have died due to progressive disease; both patients had biallelic inactivation of TP53 . Conclusion: Somatic TP53 mutations occur concurrently with both MYD88 and CXCR4 mutations in WM patients and appear to confer an aggressive disease course. Our findings highlight the need to understand the cell-specific origin of mutations detected by clinical NGS assays. DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

LINC01133 promotes hepatocellular carcinoma progression by sponging miR-199a-5p and activating annexin A2.

BackgroundLong noncoding RNAs (lncRNAs) are functionally associated with cancer development and progression. Although gene copy number variation (CNV) is common in hepatocellular carcinoma (HCC), it is not known how CNV in lncRNAs affects HCC progression and recurrence. We aimed to identify a CNV‐related lncRNA involved in HCC progression and recurrence and illustrate its underlying mechanisms and prognostic value.MethodsWe analyzed the whole genome sequencing (WGS) data of matched cancerous and noncancerous liver samples from 49 patients with HCC to identify lncRNAs with CNV. The results were validated in another cohort of 238 paired HCC and nontumor samples by TaqMan copy number assay. We preformed Kaplan‐Meier analysis and log‐rank test to identify lncRNA CNV with prognostic value. We conducted loss‐ and gain‐of‐function studies to explore the biological functions of LINC01133 in vitro and in vivo. The competing endogenous RNAs (ceRNAs) mechanism was clarified by microRNA sequencing (miR‐seq), quantitative real‐time PCR (qRT‐PCR), western blot, and dual‐luciferase reporter assays. We confirmed the binding mechanism between lncRNA and protein by RNA pull‐down, RNA immunoprecipitation, qRT‐PCR, and western blot analyses.ResultsGenomic copy numbers of LINC01133 were increased in HCC, which were positively related with the elevated expression of LINC01133. Increased copy number of LINC01133 predicted the poor prognosis in HCC patients. LINC01133 overexpression in HCC cells promoted proliferation and aggressive phenotypes in vitro, and facilitated tumor growth and lung metastasis in vivo, whereas LINC01133 knockdown had the opposite effects. LINC01133 sponged miR‐199a‐5p, resulting in enhanced expression of SNAI1, which induced epithelial‐to‐mesenchymal transition (EMT) in HCC cells. In addition, LINC01133 interacted with Annexin A2 (ANXA2) to activate the ANXA2/STAT3 signaling pathway.ConclusionsLINC01133 promotes HCC progression by sponging miR‐199a‐5p and interacting with ANXA2. LINC01133 CNV gain is predictive of poor prognosis in patients with HCC.

Copy number variations in BOLA-DQA2, BOLA-DQB, and BOLA-DQA5 show the genomic architecture and haplotype frequency of major histocompatibility complex class II genes in Holstein cows.

Bovine major histocompatibility complex (MHC) class II region contains many genes. The bovine leukocyte antigen (BoLA)-DRB3 was reportedly associated with susceptibility of various phenotypes of infections including bovine leukemia virus-induced lymphoma. However, the association of the remaining genes with various phenotypes has not been clarified due to the complicated genomic structure of the MHC class II region. Thus, in this study, we elucidated the MHC class II genomic structure, including the novel alleles and copy number variations (CNVs). We determined the copy numbers of BOLA-DQA2 (DQA2), BOLA-DQB (DQB2), BOLA-DQA5 (DQA5), BLA-DQB (DQB1), LOC100848815 (DQA1), and BOLA-DRB3 (DRB3) in 127 unrelated Holstein cows by TaqMan copy number assay. The genomes were sequenced using target next-generation sequencing (NGS) based on multiplex polymerase chain reaction. Combining the results of the copy numbers and alleles, we identified the BoLA alleles directly without haplotype estimation. Pairwise linkage disequilibrium (LD) analysis between alleles and genes were performed. The CNVs of DQA2, DQB2, and DQA5 in Holstein cows were detected. The frequency of the whole gene deletion in DQA2, DQB2, and DQA5 was 35.4%, 93.7%, and 93.7%, respectively. After target NGS, we identified 37 alleles in the six genes. Fifteen novel alleles (40.5%) were not registered in the IPD-MHC Database. LD analysis showed strong LD among the DQB2*deletion, DQA5*deletion, and DRB3*27:03 alleles. Our findings will provide important insights into the identification of the BoLA genes associated with various infection-related phenotypes.

Functional Characterization ofPsGPDin Drought Stress Response Using RNA-Seq Analysis of Transgenic Rice Plant

Plants are often exposed to biotic and abiotic stresses that affect plant growth, development, and productivity. Drought is an important abiotic stress that has a particularly serious impact on plant growth and development. We transformed rice with PsGPD using Agrobacterium-mediated transformation. We generated independent PsGPD-homozygous transgenic rice plants selected as single copy/intergenic lines by the TaqMan copy number assay and by T-DNA flanking sequences. These transgenic rice plants showed improvement of drought tolerance compared to wild-type plants under drought condition. RNA sequencing analysis showed that 2,992 genes were transcriptionally affected by the PsGPD transgene or drought treatment. In total, 145 genes were modulated by the PsGPD transgene before and after drought treatment. Among these candidate genes, 4 were up- and downregulated in all four comparisons. Several genes, including Os04t0576900, Os03t0629800, and Os04t0518400 (OsPAL7), were involved in tetrapyrrole synthesis. Os09t0522200 (DREB1A), an important component in hormone signal transduction, is a transcription factor (TF) gene that plays vital roles in stress responses. We partially characterized the functions of PsGPD in the drought stress response and the role of major TFs in the drought tolerance mechanism. These genes will be useful targets for both future research and the breeding of drought tolerance in rice.

Genetic Aberrations in the CDK4 Pathway Are Associated with Innate Resistance to PD-1 Blockade in Chinese Patients with Non-Cutaneous Melanoma.

PD-1 checkpoint blockade immunotherapy induces long and durable response in patients with advanced melanoma. However, only a subset of patients with melanoma benefit from this approach. The mechanism triggering the innate resistance of anti-PD-1 therapy remains unclear.Experimental Design: Whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) analyses were performed in a training cohort (n = 31) using baseline tumor biopsies of patients with advanced melanoma treated with the anti-PD-1 antibody. Copy-number variations (CNVs) for the genes CDK4, CCND1, and CDKN2A were assayed using a TaqMan copy-number assay in a validation cohort (n = 85). The effect of CDK4/6 inhibitors combined with anti-PD-1 antibody monotherapy was evaluated in PD-1-humanized mouse (C57BL/6-hPD-1) and humanized immune system (HIS) patient-derived xenograft (PDX) models. WES revealed several significant gene copy-number gains in the patients of no clinical benefit cohort, such as 12q14.1 loci, which harbor CDK4. The association between CDK4 gain and innate resistance to anti-PD-1 therapy was validated in 85 patients with melanoma (P < 0.05). RNA-Seq analysis of CDK4-normal cell lines and CDK4-normal tumors showed altered transcriptional output in TNFα signaling via NF-κB, inflammatory response, and IFNγ response gene set. In addition, CDK4/6 inhibitor (palbociclib) treatment increased PD-L1 protein levels and enhanced efficacy (P < 0.05) in the C57BL/6-hPD-1 melanoma cell and the HIS PDX model. In summary, we discovered that genetic aberrations in the CDK4 pathway are associated with innate resistance to anti-PD-1 therapy in patients with advanced melanoma. Moreover, our study provides a strong rationale for combining CDK4/6 inhibitors with anti-PD-1 antibody for the treatment of advanced melanomas.

MAZ promotes prostate cancer bone metastasis through transcriptionally activating the KRas-dependent RalGEFs pathway

BackgroundClinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. Myc-associated zinc-finger protein (MAZ) is a well-documented oncogene involved in the progression and metastasis of multiple cancer types, even in PCa. However, the clinical significance and biological roles of MAZ in bone metastasis of PCa remain unclear.MethodsMAZ expression was examined in PCa tissues with bone metastasis, PCa tissues without bone metastasis and metastatic bone tissues by real-time PCR and immunohistochemistry (IHC), respectively. Statistical analysis was performed to evaluate the clinical correlation between MAZ expression and clinicopathological features and bone metastasis-free survival in PCa patients. Biological roles of MAZ in bone metastasis of PCa were investigated both in vitro by transwell assay, and in vivo by a mouse model of left cardiac ventricle inoculation. The bioinformatics analysis, western blot, pull-down assays, chromatin immunoprecipitation (ChIP) and luciferase reporter assays were applied to demonstrate and examine the relationship between MAZ and its potential downstream signalling pathway. TaqMan copy number assay was performed to identify the underlying mechanism responsible for MAZ overexpression in PCa tissues.ResultsMAZ expression is elevated in PCa tissues with bone metastasis compared with that in PCa tissues without bone metastasis, and is further increased in metastatic bone tissues. High expression of MAZ positively correlates with poor overall and bone metastasis-free survival in PCa patients. Upregulating MAZ elevates, while silencing MAZ represses the invasion and migration abilities of PCa cells in vitro and bone metastasis ability in vivo. Our results further reveal that MAZ promotes bone metastasis of PCa dependent on KRas signalling, although MAZ transcriptionally upregulates KRas and HRas expression, where the Ral guanine nucleotide exchange factor (RalGEF) signaling is responsible for the different roles of KRas and HRas in mediating the pro-bone metastasis of MAZ in PCa. Finally, our results indicate that recurrent gains contribute to MAZ overexpression in a small portion of PCa tissues.ConclusionThese results indicate that the MAZ/Kras/ RalGEF signalling axis plays a crucial role in promoting PCa cell bone metastasis, suggesting a potential therapeutic utility of MAZ in bone metastasis of PCa.

454 Occurrence of TRKAIII splice variant in melanoma: a pilot study

TrkA is a member of the transmembrane neurotrophin receptor family with a critical role in cell proliferation, survival and differentiation. TrkA oncogenic activation is an emerging driver in a wide variety of solid tumors, and TrkA-targeted therapies using inhibitors have been reported to exhibit marked and durable efficacy. An alternative TrkA splice variant, the TrkAIII, characterized by exon 6-7 skipping, exhibits spontaneous aberrant activation. We aimed to explore a potential oncogenic role for TrkA in cutaneous melanoma. Cutaneous melanoma samples from patients of stage III-IV have been collected from the Department of Dermatology of University of L’Aquila. DNA and RNA was extracted from FFPE sections. The cDNA was amplified using TrkA-specific primers. The copy number variation of TrkA gene was evaluated by TaqMan Copy Number assays. Somatic mutations in the region spanning TrkA intron/exon 6 to the intron/exon 8 were analyzed by Sanger Sequencing. Overall, we collected 30 primary melanomas tissue and 12 normal skin specimens. We identified predominant alternative TrkAIII splice variant in 16 of 30 (53%) primary melanomas, while the remaining 14 (47%) cases express mainly the full length TrkA. Normal skin samples exhibit low to undetectable full length TrkA expression. The presence of TrkAIII was always confirmed by sequencing. Somatic DNA mutations in intron/exon boundary that could explain the exons skipping were not found. TrkA gene amplification was detected in 4 of 30 (13%) melanomas. Our results could open the way to new personalized therapeutic approaches for melanoma patients by targeting TrkA oncogenic signals.

Spectrum of TERT promoter mutations and mechanisms of activation in thyroid cancer.

BackgroundReactivation of telomerase reverse transcriptase (TERT) is an important event in cancer. Two hotspot mutations in the TERT promoter region, c.‐124C > T (C228T) and c.‐146C > T (C250T), occur in various cancer types including thyroid cancer. They generate de novo binding sites for E‐twenty‐six (ETS) transcription factors causing increased TERT transcription. The aim of this study was to search for novel TERT promoter mutations and additional mechanisms of TERT activation in thyroid cancer.MethodsWe studied 198 papillary thyroid carcinomas (PTCs), 34 follicular thyroid carcinomas (FTCs), 40 Hürthle cell carcinomas (HCCs), 14 poorly differentiated/anaplastic thyroid carcinomas (PDTC/ATC), and 15 medullary thyroid carcinomas (MTCs) for mutations in an −424 bp to +64 bp region of TERT. The luciferase reporter assay was used to functionally characterize the identified alterations. Copy number variations (CNVs) in the TERT region were analyzed using TaqMan copy number assay and validated with fluorescence in situ hybridization (FISH).ResultsWe detected the hotspot c.‐124C > T and c.‐146C > T mutations in 7% PTC, 18% FTC, 25% HCC, and 86% PDTC/ATC. One PTC carried a c.‐124C > A mutation. Furthermore, we identified two novel mutations resulting in the formation of de novo ETS‐binding motifs: c.‐332C > T in one MTC and c.‐104_‐83dup in one PTC. These genetic alterations, as well as other detected mutations, led to a significant increase in TERT promoter activity when assayed using luciferase reporter system. In addition, 5% of thyroid tumors were found to have ≥3 copies of TERT.ConclusionsThis study confirms the increased prevalence of TERT promoter mutations and CNV in advanced thyroid cancers and describes novel functional alterations in the TERT gene promoter, including a point mutation and small duplication. These mutations, as well as TERT copy number alterations, may represent an additional mechanism of TERT activation in thyroid cancer.

E2F1 germline copy number variations and melanoma susceptibility

BackgroundMelanoma is an aggressive type of skin cancer whose aetiology remains elusive as both environmental and genetic factors can contribute to its development. Recent studies have demonstrated the existence of multiple copies of E2F1 gene in melanoma specimens which could explain the deregulated E2F1 activity in this type of cancer. This finding suggests a key role for this transcription factor in the malignant transformation of melanocytes. Therefore, E2F1 has been considered as a potential therapeutic target for this form of skin cancer. Since germline copy number variations (CNVs) have been associated with increased susceptibility to different types of cancer, the aim of our study was to assess germline E2F1 CNV in melanoma patients. However, CNVs not necessarily lead to gene dosage imbalance, hence, further factors, in association with CNVs, could contribute to clinical manifestations. Considering that heat stress has been hypothesised as a contributing factor to skin cancer, we also investigated the effect of heat stress on E2F1 expression.MethodsE2F1 CNV was measured in genomic DNA isolated from blood of 552 patients diagnosed with melanoma and 520 healthy subjects using TaqMan Copy Number Assays. E2F1 mRNA expression was also evaluated by RT-qPCR in the melanoma cell line, SK MEL 267, before and after exposure to heat stress.ResultsWe found that patients diagnosed with melanoma (1.6%, 9/552) harboured frequently altered germline E2F1 copies compared to healthy subjects (0%, 0/520). Moreover, the difference among the two groups was statistically significant (p = 0.004). Furthermore, we found that heat exposure alone can significantly induce E2F1 expression.ConclusionsThis is the first study that shows a relation between germline E2F1 CNV and melanoma, suggesting that altered copies of this gene might be a predisposing factor to skin cancer. Our results also suggest that environmental insults, such as heat stress, could contribute to an aberrant E2F1 activity by inducing E2F1 mRNA expression. Therefore, subjects with multiple constitutive copies of E2F1 are at greater risk of developing melanoma when exposed to heat. Altogether our results corroborate with the hypothesis that susceptibility to melanoma depends on both the environment and genetic factors.

The association between methylation patterns of DNAH17 and clinicopathological factors in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is a malignancy with poor prognosis. Complex genetic and epigenetic alterations are the two primary causes of HCC. The aim of the study was mainly to explore the correlation between the methylation status of DNAH17 and HCC.MethodsWe evaluated the methylation levels of DNAH17 in 163 HCC samples and their paired normal tissue using Sequenom EpiTYPER assays and performed the TaqMan copy number assay to assess the copy number status of DNAH17 in HCC samples.ResultsThe mean methylation levels were significantly decreased in the tumor tissues compared to the paired normal tissues in both selected regions of DNAH17 (amplicon 1:58.7% vs 84.5%, P < 0.0001; amplicon 2:69.9% vs 84.5%, P = 0.0060). Contrarily，both RNA‐seq and immunohistochemistry indicated the expression of DNAH17 was increased in tumor tissues (P < 0.05). DNMT inhibitor decitabine treatment could increase the expression of DNAH17 in HCC cell lines. DNAH17 gene amplification always companied with hypomethylation status. Moreover, hypomethylation status was associated with several clinical characteristics, such as male patients, higher AFP values, higher age of onset, fibrous capsules, tumor necrosis, liver cirrhosis, and tumor thrombus (P < 0.05). Receiver operator characteristic (ROC) curve analysis demonstrated the methylation levels of DNAH17 could efficiently predict the existence of the fibrous capsule (AUC = 0.695) and tumor thrombus (AUC = 0.806).ConclusionsThese findings suggested that aberrant methylation of DNAH17 was associated with comprehensive HCC clinicopathological factors and could be a promising biomarker for tumor thrombosis in HCC patients.

Expression and copy number gains of the RET gene in 631 early and mid stage non-small cell lung cancer cases.

BackgroundTo identify whether RET is a potential target for NSCLC treatment, we examined the status of the RET gene in 631 early and mid stage NSCLC cases from south central China.Methods RET expression was identified by Western blot. RET‐positive expression samples were verified by immunohistochemistry. RET gene mutation, copy number variation, and rearrangement were analyzed by DNA Sanger sequencing, TaqMan copy number assays, and reverse transcription‐PCR. ALK and ROS1 expression levels were tested by Western blot and EGFR mutation using Sanger sequencing.ResultsThe RET‐positive rate was 2.5% (16/631). RET‐positive expression was related to poorer tumor differentiation (P < 0.05). In the 16 RET‐positive samples, only two samples of moderately and poorly differentiated lung adenocarcinomas displayed RET rearrangement, both in RET‐KIF5B fusion partners. Neither ALK nor ROS1 translocation was found. The EGFR mutation rate in RET‐positive samples was significantly lower than in RET‐negative samples (P < 0.05).Conclusion RET‐positive expression in early and mid stage NSCLC cases from south central China is relatively low and is related to poorer tumor differentiation. RET gene alterations (copy number gain and rearrangement) exist in all RET‐positive samples. RET‐positive expression is a relatively independent factor in NSCLC patients, which indicates that the RET gene may be a novel target site for personalized treatment of NSCLC.

Abstract B15: Copy number and mRNA expression alterations of the PI3K/AKT and inflammatory pathways in penile cancer patients

Abstract Penile cancer (PeCa) is a rare neoplasm in developed countries; however, its incidence is high in developing countries. In Brazil, the North and Northeast regions present the highest incidence of cases; in the state of Maranhão, in particular, about four penectomy procedures are conducted weekly in patients with advanced PeCa. Several risk factors have been attributed to the development of PeCa, including infection with human papillomavirus (HPV) and/or chronic inflammation. However, the genetic pathways that are involved in the malignant transformation impacted by these factors are not completely known. Gene alterations affecting the PI3K/AKT signaling pathway are commonly observed in many human cancers. Mutations in the PI3KCA gene and overexpression of EGFR are described in PeCa; however, their prognostic value and/or association with HPV and inflammatory markers are not well known. Therefore, in this study our objectives were to investigate copy number alterations (CNAs) in specific genes involved in the PI3K/AKT pathway (PIK3CA, AKT2 and PTEN) and the expression of the inflammatory associated markers COX2 and EGFR and their association with HPV infection and histopathologic parameters from the patients. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from 37 patients with penile cancer were obtained from Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) and Hospital Universitário Presidente Dutra (HU-UFMA), São Luis, Maranhão. The samples were obtained at the time of the surgery; 78.9% and 21.1% of the patients underwent partial and total penectomy, respectively. Lymphadenectomy was conducted in 76.7% of the patients. The average age of the patients was 59.9+18.3 years and the average tumor size was 4.44+2.39 mm. Most of the cases were of the spinocellular carcinoma histology (48.6%), followed by epidermoid (40.5%) and squamous cell carcinoma (10.8%). Tumor grade II was observed in 62.2% of the patients, and grades I and III in 19% each. Lymphatic and perineural invasion was observed in 33.3% and 42% of the patients, respectively. DNA and RNA were isolated from the tumor tissue samples using AllPrep DNA/RNA FFPE Kit. Nested PCR genotyping assay was carried out to determine HPV subtypes of the patients. Copy number of the PIK3CA, AKT 2, and PTEN genes were accessed by TaqMan copy number assays and mRNA expression of the inflammatory associated genes EGFR and COX2 by qRT-PCR analysis. HPV infection was detected in 91.9% of the patients for 14 subtypes of HPV (6, 16, 30, 35, 44, 51, 52, 53, 56, 59, 66, 70, 73, and 74), 23.5% of whom presented with multiple HPV infection. The most frequent subtypes observed, affecting 34.4% of the patients, were the subytpes 16 and 18. Deletion of copy number was observed for all three genes analyzed in almost all the patients (90%); PIK3CA presented the highest frequency of homozygous deletion (56.3%), followed by PTEN (39.4%) and AKT2 (36.4%). No gains and/or amplifications on these genes were observed in any of the cases analyzed. EGFR and COX2 genes were observed to be overexpressed in 87% and 40% of the tumors, respectively. No association was observed between copy number and gene expression alterations of the genes analyzed and HPV infection, and the clinical-histopathologic parameters analyzed. However, patients infected with the HPV subtypes 16 and 18 presented tumors of more advanced grade (grade III). Homozygous deletion of the PIK3CA, PTEN, and AKT2 as well as overexpression of COX2 and EGFR genes were observed in the group of PeCa patients from the state of Maranhão. Although a remarkably high level of patients presented with HPV infection (92%), these genomic alterations occurred independently of this infection and of the other prognostic parameters evaluated. These findings support the involvement of alterations in the PI3K/AKT and inflammatory pathways in the etiology of PeCa, and open up opportunities of using the available or novel therapeutic strategies targeting these pathways for PeCa patients. Citation Format: Silma Regina Ferreira Pereira, Juliana Melo Macedo, Marta Regina de Castro Belfort, Elis Vanessa de Lima Silva, Leudivan Ribeiro Nogueira, José Ribamar Calixto, Ronald Coelho, Antonio Augusto Lima Teixeira Júnior, Jaqueline Diniz Pinho, André Salim Khayat, Gyl Eanes Barros Silva, Luciane Regina Cavalli. Copy number and mRNA expression alterations of the PI3K/AKT and inflammatory pathways in penile cancer patients [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B15.

Circulating androgen receptor combined with 18F-fluorocholine PET/CT metabolic activity and outcome to androgen receptor signalling-directed therapies in castration-resistant prostate cancer

The association between choline uptake and androgen receptor (AR) expression is suggested by the upregulation of choline kinase-alpha in prostate cancer. Recently, detection of AR aberration in cell-free DNA as well as early 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) were associated with outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone and enzalutamide. We aimed to make a direct comparison between circulating AR copy number (CN) and choline uptake at FCH-PET/CT. We analysed 80 mCRPC patients progressing after docetaxel treated with abiraterone (n = 47) or enzalutamide (n = 33). We analysed AR CN from plasma samples using digital PCR and Taqman CN assays and total lesion activity (TLA) and metabolic tumor volume (MTV) on FCH-PET/CT at baseline. A meaningful correlation was showed among AR gain and TLA/MTV compared to AR non-gained cases (P = 0.001 and P = 0.004, respectively), independently from type of treatment. Multivariate analysis revealed that AR CN and only TLA were associated with both shorter PFS (P < 0.0009 and P = 0.026, respectively) and OS (P < 0.031 and P = 0.039, respectively). AR gain appeared significantly correlated with choline uptake represented mainly by TLA. Further prospective studies are warranted to better address this pathway of AR-signalling and to identify multiplex biomarker strategies including plasma AR and FCH-PET/CT in mCRPC patients.

Deletions at SLC18A1 increased the risk of CRC and lower SLC18A1 expression associated with poor CRC outcome.

Copy number variations (CNVs) contribute to the development of colorectal cancer (CRC). We conducted a two-stage association study to identify CNV risk loci for CRC. We performed a gene-based rare CNV study on 694 sporadic CRC and 1641 controls using Illumina Human-OmniExpress-12v1.0 BeadChips, and further replicated in 934 CRC cases and 2680 controls for risk CNVs by using TaqMan Copy Number Assay. Tumor buddings, cancer cells in the center of primary tumor and normal intestinal epithelial cells were captured using laser capture microdissection (LCM) and were assayed using AffymetrixGeneChip® Human Genome U133 Plus 2.0 Array. In addition, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus data were assessed for the effects of risk CNVs. We found that germline deletions affecting the last six exons of SLC18A1 significantly associated with CRC with a combined P value of 6.4 × 10-5 by a two-stage analysis. Both in TCGA CRC RNA seq dataset and GDS4382, SLC18A1 was significantly down regulated in CRC tissues than in paired normal tissues (N = 32 and 17 pairs, P = 0.004 and 0.009, respectively). In LCM samples, similar observations were obtained that the expression levels of SLC18A1 in the tumor buddings, cancer cells in the center of primary tumor, and stroma of both tumor budding and cancer cells were lower than normal intestinal epithelial and stromal cells (fold change = 0.17-0.62, 0.12-0.57 and 0.37-0.68, respectively). In summary, the germline deletions at SLC18A1 contributed to the development of CRC. The role of SLC18A1 required further exploration.