Co-amplification Of Genes Research Articles

A PET-Surrogate Signature for the Interrogation of the Metabolic Status of Breast Cancers.

Metabolic alterations in cancers can be exploited for diagnostic, prognostic, and therapeutic purposes. This is exemplified by 18F-fluorodeoxyglucose (FDG)-positron emission tomography (FDG-PET), an imaging tool that relies on enhanced glucose uptake by tumors for diagnosis and staging. By performing transcriptomic analysis of breast cancer (BC) samples from patients stratified by FDG-PET, a 54-gene signature (PETsign) is identified that recapitulates FDG uptake. PETsign is independently prognostic of clinical outcome in luminal BCs, the most common and heterogeneous BC molecular subtype, which requires improved stratification criteria to guide therapeutic decision-making. The prognostic power of PETsign is stable across independent BC cohorts and disease stages including the earliest BC stage, arguing that PETsign is an ab initio metabolic signature. Transcriptomic and metabolomic analysis of BC cells reveals that PETsign predicts enhanced glycolytic dependence and reduced reliance on fatty acid oxidation. Moreover, coamplification of PETsign genes occurs frequently in BC arguing for their causal role in pathogenesis. CXCL8 and EGFR signaling pathways feature strongly in PETsign, and their activation in BC cells causes a shift toward a glycolytic phenotype. Thus, PETsign serves as a molecular surrogate for FDG-PET that could inform clinical management strategies for BC patients.

The genomic landscape and prognostic significance of 11.q.13 amplification in breast, head & neck, and lung cancer.

e18037 Background: The 11.q.13 (11q) chromosomal band is amplified in many cancers and is associated with poor outcomes in Head & Neck (HN) cancer. The 11q amplicon contains several putative oncogenes, but it’s unclear which amplified genes drive clinical outcomes. We compared the genomic landscape and prognostic significance of 11q amplification in Breast (BC), HN, and non-small cell lung cancer (NSCLC) tumors and identify genes whose expression may be driving clinical outcomes. Methods: DNA (592 genes or WES)/RNA (WTS) sequencing was performed for BC (N = 6,967), HN (N = 2,337), and NSCLC (N = 13,433) tumors submitted to Caris Life Sciences (Phoenix, AZ). 11q amplification (CNA) was defined as tumors with concurrent copy number amplification with copies of FGF3, FGF4, FGF19, and CCND1. Non-amplified tumors (copy number normal, CNN) were defined as a < 4 copies for all four genes. Her2/Neu (+: ≥3+ and > 10%) and HR (ER or PR+: ≥1+ and ≥1%) expression was tested by IHC. tests were applied as appropriate, with P-values adjusted for multiple comparisons. Real-world overall survival (OS) data was obtained from insurance claims, and Kaplan-Meier estimates were calculated for a molecularly defined subset of tumors. 11q genes that were two-fold over expressed in CNA vs CNN were further investigated. The hazard ratio (HR) was calculated for subgroups stratified by median expression of 11q genes. The genes with the five largest HRs (worse OS for CNA, p < .05) were identified as potential driver genes. Results: 11q CNA was observed in 10% of BC, 12% of HN, and 3% of NSCLC. 11q CNA rates were enriched in HR+ BC (HR+/HER2-: 17%, HR+/HER2+: 19%) as compared to HR- tumors (HR-/HER2+: 6%, TNBC: 3%, p < .05). TP53 mutations were less frequent in CNA BC (32 vs 53% CNN) but more frequent in CNA HN (91 vs 52%) and NSCLC (89 vs 66%, p < .05 all). In NSCLC, CNA tumors had a lower rate of EGFR (4.3 vs 12%), KRAS (6.7 vs 28%) and STK11 (5.7 vs 13%) mutations ( p < .05). Over 34 gene were amplified as compared to CNN ( > 3% increase, p < .05), including FGFR3 (11 vs 0.2%) and PDCD1 (10 vs 0). MEN1 (5 vs 0%) was the only 11q gene amplified. CNA was associated with worse OS in HR-/HER2+ (HR 6.3 [2.6-15.5], p = .001), while no difference in OS was observed in BC (HR 1.0 [.9-1.1], p = .62). Compared to CNN, CNA had worse OS in HN (HR 1.5 [1.3-1.7], p < .001) and NSCLC (HR 1.3 [1.2-1.4], p < .001). Stratification by 11q gene expression identified DHCR7, GAL, ANO1, MRPL21, and FADD as the strongest predictors of OS in HN tumors, while GAL, MYEOV, MRPL21, OVOL1, and DHCR7 were strongest in NSCLC (HR: 1.3-1.2). Conclusions: 11q CNA shows differential association with key driver mutations across cancer types that provides new insight to patient subpopulations harboring these alterations. Co-amplification of non-11q genes in NSCLC suggests a unique mechanism of genome instability. The role of DHCR7 and GAL as predictors of clinical outcomes in HN and NSCLC CNA tumors warrants further investigation.

Metastatic Prostate Cancers with BRCA2 versus ATM Mutations Exhibit Divergent Molecular Features and Clinical Outcomes.

In patients with metastatic prostate cancer (mPC), ATM and BRCA2 mutations dictate differences in PARPi inhibitor response and other therapies. We interrogated the molecular features of ATM- and BRCA2-mutated mPC to explain the divergent clinical outcomes and inform future treatment decisions. We examined a novel set of 1,187 mPCs after excluding microsatellite-instable (MSI) tumors. We stratified these based on ATM (n = 88) or BRCA2 (n = 98) mutations. As control groups, mPCs with mutations in 12 other homologous recombination repair (HRR) genes were considered non-BRCA2/ATM HRR-deficient (HRDother, n = 193), whereas lack of any HRR mutations were considered HRR-proficient (HRP; n = 808). Gene expression analyses were performed using Limma. Real-world overall survival was determined from insurance claims data. In noncastrate mPCs, only BRCA2-mutated mPCs exhibited worse clinical outcomes to AR-targeted therapies. In castrate mPCs, both ATM and BRCA2 mutations exhibited worse clinical outcomes to AR-targeted therapies. ATM-mutated mPCs had reduced TP53 mutations and harbored coamplification of 11q13 genes, including CCND1 and genes in the FGF family. BRCA2-mutated tumors showed elevated genomic loss-of-heterozygosity scores and were often tumor mutational burden high. BRCA2-mutated mPCs had upregulation of cell-cycle genes and were enriched in cell-cycle signaling programs. This was distinct from ATM-mutated tumors. Tumoral ATM and BRCA2 mutations are associated with differential clinical outcomes when patients are stratified by treatments, including hormonal or taxane therapies. ATM- and BRCA2-mutated tumors exhibited differences in co-occurring molecular features. These unique molecular features may inform therapeutic decisions and development of novel therapies.

39. Associations between somatically altered genes and recurrence outcomes in estrogen receptor positive breast cancer

The relationships between somatic mutations, copy number alterations (CNA) and outcomes in estrogen receptor positive (ER+) breast cancer is understudied. We have earlier published associations between rare and low frequency mutations and clinical outcomes in ER+ disease (PMID:30181556). As an extension of the study, FFPE blocks were obtained from 889 additional patients from the TransATAC study, an Anastrozole (A) or Tamoxifen (T) alone or combined randomized trial with 10-year follow-up. Tumor samples were reviewed, DNA isolated, and targeted sequencing performed for 83 genes. Mutation calling methods were previously reported. Average Copy number (CN) ratios were estimated for 875 tumors and 94 unmatched normal breast tissues using CNVkit. 841 samples met minimum quality and coverage criteria. For a given gene, gains and losses were defined as tumor CN-ratio above or below 95% confidence-interval of the distribution of normal CN-ratios. Hazard ratio was calculate for recurrence free survival (RFS) for somatically altered genes. TP53 mutations was one of the most important prognostic markers in this cohort. Non-silent mutations in ATM, ERBB3, ERBB4, KMT2C and NF1 associated with poor RFS. Co-deletion and co-amplification of genes respectively on chromosomal region 1p (MAGI3, JAK1, ARID1A, MTOR) and 20q13 (MYBL2, NCOA3, AURKA) associated with poor RFS. Deletion of FZD7 and PPP2R2A and amplification of MAP3K4, MAP3K13, LYN and BRAF associated with poor RFS. In summary, we present the prognostic role of multiple recurrently altered genes and their association with poor RFS in ER+ breast cancer. This warrants further investigation within clinical subtypes and treatment-specific sub-populations.

Abstract 1882: Passenger gene co-amplification leads to collateral vulnerabilities in cancer

Abstract The activation of oncogenes via genomic amplification is a hallmark of the cancer genomes, yet such amplifications frequently also encompass neighboring genes. Such passenger genes are involved in essential cellular activities. We hypothesized that co-amplification of passenger genes create cancer-specific collateral therapeutic vulnerabilities. To test this, we analyzed one of the most commonly amplified gene locus near MYCN, which is amplified in nearly 25% of neuroblastoma and around 5% of all cancers. We observed that DDX1, a DEAD-Box helicase, is the most frequently co-amplified passenger gene in MYCN-amplified cancers. Using CRISPR/Cas9 loss-of-function screen results from the Cancer Dependency Map across 739 human cancer cell lines, we reveal that cells with DDX1 co-amplification are highly dependent on the mTOR complex 1(mTORC1). Mechanistically, western blot and immunoprecipitation analyses shows that DDX1 expression leads to higher mTORC1 pathway activity through the interaction between DDX1 and alpha-ketoglutarate dehydrogenase(alpha-KGDH) in mitochondria. DDX1 hijacks alpha-KGDH complex, thereby impairing the function of the Krebs cycle by interfering with the conversion of alpha-ketoglutarate to succinyl-coA. Live cell metabolomics shows accumulated alpha-ketoglutarate in turn activates mTORC1 pathway to promote the ATP production via oxidative phosphorylation(OXPHOS) to maintain cell survival. Consequently, interference with mTORC1 function reduced ATP production and led to accumulated alpha-KG leading to cell death, specifically in DDX1 expressing cells. This suggests that co-amplification of a passenger gene can result in collateral vulnerabilities in cancers, which has the potential to transform future therapeutic target discovery in oncology. Citation Format: Yi Bei, Marieluise Kirchner, Luca Brame, Raphaela Fritsche-Guenther, Julia Koeppke, Anja Heeren-Hagemann, Philipp Mertins, Jan Dörr, Anton George Henssen. Passenger gene co-amplification leads to collateral vulnerabilities in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1882.

Clinical Impact of 11q13.3 Amplification on Immune Cell Infiltration and Prognosis in Breast Cancer.

IntroductionAmplification of the 11q13.3 locus has been observed in various tumors. This study sought to determine the correlation of gene amplification at the 11q13.3 locus with the immune status and survival of breast cancer.MethodsAmplification of the 11q13.3 locus was characterized by analyzing a publicly available database from the cBioPortal platform (TCGA). The correlation of amplified genes with immune cell infiltration in breast cancer was further analyzed using the TIMER2.0 platform. Immunohistochemical staining was used to determine the expression levels of Cyclin D1 (CCND1), Fas-associated death domain (FADD) and P53 in 156 clinical breast cancer samples.ResultsThis study revealed that amplification of the 11q13.3 amplicon in breast cancer is likely more frequently detected in luminal B breast cancer. Moreover, high expression or amplification of CCND1, fibroblast growth factor 3 (FGF3), fibroblast growth factor 4 (FGF4), fibroblast growth factor 19 (FGF19) and FADD was inversely correlated with the abundance of CD4+ T cells and dendritic cell infiltration in breast cancer (P < 0.05). Data analysis also demonstrated that high expression of CCND1, FGF4 and FADD mRNA levels was closely correlated with shorter recurrence-free survival (RFS) in patients with breast cancer (P < 0.05). The results of immunohistochemical staining from clinical samples further confirmed that high expression of CCND1 and FADD was frequently detected in luminal B and high-grade breast cancer with shorter metastasis-free survival times (P < 0.05).ConclusionThis study demonstrated that coamplification of genes located on the 11q13.3 amplicon is frequently detected in luminal B subtype breast cancer and is closely associated with worse survival in patients with breast cancer. Moreover, coamplification of the CCND1-FGF locus might decrease antitumor immune activity in breast cancer, indicating that coamplification of the 11q13.3 amplicon is likely to be a key determinant of therapeutic resistance and accelerate the aggressive evolution of breast cancer.

Heat Shock Proteins and HSF1 in Cancer.

Fitness of cells is dependent on protein homeostasis which is maintained by cooperative activities of protein chaperones and proteolytic machinery. Upon encountering protein-damaging conditions, cells activate the heat-shock response (HSR) which involves HSF1-mediated transcriptional upregulation of a group of chaperones – the heat shock proteins (HSPs). Cancer cells experience high levels of proteotoxic stress due to the production of mutated proteins, aneuploidy-induced excess of components of multiprotein complexes, increased translation rates, and dysregulated metabolism. To cope with this chronic state of proteotoxic stress, cancers almost invariably upregulate major components of HSR, including HSF1 and individual HSPs. Some oncogenic programs show dependence or coupling with a particular HSR factor (such as frequent coamplification of HSF1 and MYC genes). Elevated levels of HSPs and HSF1 are typically associated with drug resistance and poor clinical outcomes in various malignancies. The non-oncogene dependence (“addiction”) on protein quality controls represents a pancancer target in treating human malignancies, offering a potential to enhance efficacy of standard and targeted chemotherapy and immune checkpoint inhibitors. In cancers with specific dependencies, HSR components can serve as alternative targets to poorly druggable oncogenic drivers.

Convergent evolution of a genomic rearrangement may explain cancer resistance in hystrico- and sciuromorpha rodents

The rodents of hystricomorpha and sciuromorpha suborders exhibit remarkably lower incidence of cancer. The underlying genetic basis remains obscure. We report a convergent evolutionary split of human 3p21.31, a locus hosting a large number of tumour-suppressor genes (TSGs) and frequently deleted in several tumour types, in hystrico- and sciuromorphs. Analysis of 34 vertebrate genomes revealed that the synteny of 3p21.31 cluster is functionally and evolutionarily constrained in most placental mammals, but exhibit large genomic interruptions independently in hystricomorphs and sciuromorphs, owing to relaxation of underlying constraints. Hystrico- and sciuromorphs, therefore, escape from pro-tumorigenic co-deletion of several TSGs in cis. The split 3p21.31 sub-clusters gained proximity to proto-oncogene clusters from elsewhere, which might further nullify pro-tumorigenic impact of copy number variations due to co-deletion or co-amplification of genes with opposing effects. The split of 3p21.31 locus coincided with the accelerated rate of its gene expression and the body mass evolution of ancestral hystrico- and sciuromorphs. The genes near breakpoints were associated with the traits specific to hystrico- and sciuromorphs, implying adaptive significance. We conclude that the convergently evolved chromosomal interruptions of evolutionarily constrained 3p21.31 cluster might have impacted evolution of cancer resistance, body mass variation and ecological adaptations in hystrico- and sciuromorphs.

Multiomic analysis to reveal distinct molecular profiles of uterine and nonuterine leiomyosarcoma.

11555 Background: Leiomyosarcoma (LMS) is a rare group of mesenchymal malignancies found in the uterus, retroperitoneum, skin, or other soft-tissue sites. Treatment for LMS is extrapolated from trials including both uterine (uLMS) and non-uLMS subtypes, although whether they respond similarly and have similar outcomes from treatment is not clear. We examined the molecular composition of LMS by site of origin to better inform future drug development and trial design. Methods: We reviewed 1115 specimens with LMS histology tested by Caris Life Sciences for targeted exome (NextSeq, 592 gene panel), whole exome, and whole transcriptome sequencing (NovaSeq). Specimens were stratified into uLMS, rpLMS (retroperitoneal), and otherLMS (non-uterine/retroperitoneal) subgroups based on tumor origin sites. Genomic data was analyzed for mutations, copy number aberrations, and fusions. RNA expression profiling included evaluation of individual genes and gene set enrichment analysis (GSEA). P-value adjustment performed by the Benjamini-Hochberg procedure. Results: The study cohort was comprised of 62.9% uLMS (n = 701), 14.9% rpLMS (n = 166) and 22.2% otherLMS (n = 248) specimens. Overall, LMS specimens most frequently harbored TP53 (64%, n = 612), ATRX (30%, n = 219), RB1 (22%, n = 156), and MED12 (16%, n = 94) mutations, with these genes accounting for 74.4% (n = 1044) of all observed pathogenic/likely pathogenic mutations. RB1 mutations were significantly less common in uLMS (15%) compared to rpLMS (30%, p &lt; 0.05) and otherLMS (33%, p &lt; 0.01), whereas MED12 mutations were almost exclusive to uLMS (22% vs 1% rpLMS, 3% otherLMS, p &lt; 0.05). MAP2K4 copy number amplification were more common in rpLMS (22%, p &lt; 0.001) and otherLMS (14%, p &lt; 0.182) compared to uLMS (7%), with frequent co-amplification of nearby genes ( FLCN, GID4, SPECC1, GAS7, PER1, and AURKB) located at chr17p11-13. Actionable gene fusions involving ALK (2.1%, n = 11), FGFR1 (0.2%, n = 1), and NTRK1/2 (0.2%, n = 1 each) were rare overall, with similar prevalence across subtypes. Genomic alteration rates were not significantly different between rpLMS and otherLMS subtypes . RNA expression profiling identified significant upregulation of PI3K/AKT/mTOR, DDR, WNT/Beta-Catenin pathway genes in non-uLMS. GSEA indicated several immune-related gene sets were enriched in rpLMS and otherLMS compared to uLMS. Conclusions: Comprehensive molecular profiling suggests that LMS originating from the uterus represents a molecularly distinct disease compared to other primary sites of origin. We identified key genomic patterns which have potential for targeted therapy. These data provide insight for the framework of future clinical trials designed to separate uLMS from non-uLMS histologies, although further subdivision does not appear to be warranted.

Clinicopathological characteristics of pulmonary artery intimal sarcoma

Objective: To describe the clinicopathological features of pulmonary artery intimal sarcoma (PAIS), and to understand its molecular alterations. Methods: Sixty cases of pulmonary artery endarterectomy performed at the China-Japan Friendship Hospital, Beijing, China from January 2017 to January 2020 were reviewed. Clinical data of 5 patients with pulmonary artery intimal sarcoma were collected. Hematoxylin-eosin staining, immunohistochemistry staining and fluorescence in situ hybridization (FISH) were performed to evaluate the pathological features. RNA sequencing was conducted to assess the fusion gene changes in PAIS. Results: The detection rate of PAIS was 8.3% (5/60), with the median age of 49 years and a female predominance. Their clinical manifestations were non-specific. Histopathological examination showed that the tumors were composed of malignant spindle or epithelioid cells, with various degrees of atypia. Focal heterologous osteosarcomatous or leiomyosarcomatous differentiation was noted. The tumor cells could express PDGFRA, CDK4 and MDM2 with co-amplification of MDM2, CDK4 and EGFR genes. RNA sequencing detected multiple in-frame fusions in the tumors. Conclusions: PAIS is a rare, highly heterogeneous, and poorly-or un-differentiated sarcoma accompanied by complex changes of multiple genes.It has no known effective treatments, and thus has a poor prognosis.

A comparative study of RTK gene status between primary tumors, lymph-node metastases, and Krukenberg tumors

Krukenberg tumor (KT) refers to a rare ovarian tumor that has metastasized from a primary site. Patients with KTs have a poorer prognosis and worse survival. Thus far, little is known about the frequency of receptor tyrosine kinase (RTK) gene amplification and the concordance of gene amplification between primary tumors, lymph-node metastases, and KTs. Herein, 50 paired samples, including primary cancers, metastatic lymph nodes, and KTs were collected, and RTK gene amplification was tested by fluorescence in situ hybridization (FISH). There were four cases positive for human epidermal growth factor receptor type 2 (HER2) amplification, all of which showed conversion of HER2 status between different lesions. Of the two cases with c-mesenchymal–epithelial transition (c-MET) amplification, the primary tumors and lymph nodes were negative while the right involved ovaries were positive. Inconsistent fibroblast growth factor receptor 2 (FGFR2) status in different lesions was observed in three of the six FGFR2-amplified cases. Co-amplification of RTK genes was identified in only one patient for primary cancer and two for KTs. Collectively, there were 46, 48, 50, and 44 cases negative for HER2, c-MET, EGFR, and FGFR2 amplification in all lesions, respectively. There was no significant difference in overall survival between KTs of gastric origin and colorectal origin. However, of all synchronous cancers, KTs of colorectal origin had a better prognosis than those of gastric origin. In conclusion, the positive rate of RTK gene amplification in KTs was low. Intratumoral heterogeneity was frequent in KTs with RTK gene amplification. A mutually exclusive pattern of RTK gene amplification was dominant in primary cancers, lymph-node metastases, and KTs. There was no survival difference between KTs of gastric origin and colorectal origin. However, of all synchronous cancers, KTs of colorectal origin had a better prognosis than those of gastric origin.

Coamplification of Mycobacterium leprae genome sections by real-time PCR: Detection of the pathogen and the possibility of a semi-quantitative assessment of the bacterial load

Aim. To develop a method for coamplification of single-copy genes and repetitive elements of the Mycobacterium leprae genome in the analysis of clinical material from leprosy patients with an assessment of the clinical significance of the study results. Materials and methods . Skin scarification and biopsy samples from patient R. with a diagnosis of “A30.5 Leprosy. Multibacillary form. Lepromatous type. Active stage” were used as empirical material for the study. A search for M. leprae DNA in the clinical material was performed by the method of real-time PCR (RT-PCR) using primers and hydrolysis probes for the single-copy species-specific genes rpoB (encodes the β -subunit of bacterial RNA polymerase), sodA (encodes the superoxide dismutase enzyme) and mntH (encodes the manganese transport protein), as well as for RLEP — the non-coding repetitive element of the genome . Results. Using various RT-PCR assays, consistent results were obtained concerning the presence or absence of M. leprae DNA in the studied clinical samples. The high sensitivity of PCR was confirmed for the detection of the repetitive element RLEP compared to the single-copy genes rpoB , sodA and mntH , which consists in reducing the number of amplification cycles (Ct) needed for exceeding the threshold fluorescence value of hydrolysis probes and leading to the maximum intensity of the fluorescence signal. When constructing standard graphs for calibrating the accumulation of a fluorescent signal for simultaneously analyzed portions of the M. leprae genome in dilutions from 1 to 1,000, significant differences in the results of co-amplification were noted depending on the quantitative presence of the DNA being detected. Conclusion. Coamplification of M. leprae genome sections with varying degrees of copy number variation by the RTPCR method provides for effective detection of the M. leprae DNA in clinical material and forms a basis for a quantitative assessment of the bacterial load in skin scarification and biopsy samples.

Genetic alterations of epithelial polarity genes are associated with loss of polarity in invasive breast cancer

Breast cancer remains a leading cause of cancer-related death for women. The stepwise development of breast cancer through preinvasive to invasive disease is associated with progressive disruption of cellular and tissue organization. Apical-basal polarity is thought to be a barrier to breast cancer development, but the extent and potential mechanisms that contribute to disrupted polarity are incompletely understood. To investigate the cell polarity status of invasive breast cancers, we performed multiplex imaging of polarity markers on tissue cores from 432 patients from a spectrum of grades, stages and molecular subtypes. Apical-basal cell polarity was lost in 100% of cells in all cases studied, indicating that loss of epithelial polarity may be a universal feature of invasive breast cancer. We then analyzed genomic events from the TCGA dataset for an 18-gene set of core polarity genes. Coamplification of polarity genes with established breast oncogenes was found, which is consistent with functional cooperation within signaling amplicons. Gene-expression levels of several polarity genes were significantly associated with survival, and protein localization of Par6 correlated with higher grade, nodal metastasis and molecular subtype. Finally, multiple hotspot mutations in protein-protein interaction domains critical for cell polarity were identified. Our data indicate that genomic events likely contribute to pervasive disruption of epithelial polarity observed in invasive breast cancer.

Non-invasive prenatal diagnosis of foetal gender through maternal circulation in first trimester of pregnancy

Owing to the lack of perfect accuracy and sufficient sample size in previously performed studies on cell-free foetal DNA (cffDNA) for detection of foetal gender through maternal plasma, this study aimed to investigate the efficiency of using two Y-chromosome specific probes in foetal sex determination during first trimester of pregnancy. Five millilitres of whole blood was drawn from 192 pregnant women (10–12 weeks) and was subjected to isolate cffDNA following separation of plasma. TaqMan Real-time PCR was performed on isolated cffDNA using primer pairs and probes specific for SRY, ZFY and β-globin genes. Co-amplification of ZFY and SRY genes was detected in 103 samples confirmed after the birth. Sensitivity and specificity of the test were calculated to be 100%. Further study on larger sample size is required to confirm the reproducibility of the present test in early and non-invasive determination of foetal sex.IMPACT STATEMENTWhat is already known on this subject? Foetal gender analysis through maternal plasma has been investigated in some cell-free foetal DNA (cffDNA) analysis. However, the detection rate and method of cffDNA analysis were different among various studies.What do the results of this study add? This study introduced a modified simple probe based real time analysis with perfect detection rate.What are the implications of these findings for clinical practice and/or further research? The proposed method can be used as diagnostic test in all laboratories around the world using real-time PCR to non-invasively determine the foetal gender in the initial weeks of pregnancy following confirmation in larger sample size.

Validation and Application of a Real-Time PCR Assay Based on the CRISPR Array for Serotype-Specific Detection and Quantification of Enterohemorrhagic Escherichia coli O157:H7 in Cattle Feces

Several real-time quantitative PCR (qPCR) assays have been developed for detection and quantification of Escherichia coli O157:H7 in complex matrices by targeting genes for serogroup-specific O-antigen ( rfbEO157), H7 antigen, and one or more major virulence factors (Shiga toxin and intimin). A major limitation of such assays is that coamplification of H7 and virulence genes in a sample does not signal association of those genes with the O157 serogroup. Clusters of regularly interspaced short palindromic repeats (CRISPR) polymorphisms are highly correlated with certain enterohemorrhagic E. coli (EHEC) serotypes, including O157:H7, and the presence of genes for Shiga toxin ( stx1 and stx2) and intimin ( eae). Our objectives were to develop and validate a qPCR assay targeting the CRISPR array for the detection and quantification of EHEC O157:H7 in cattle feces and to evaluate the applicability of the assay for detection of and comparison with a four-plex qPCR assay targeting rfbEO157, stx1, stx2, and eae genes and a culture method. Detection limits of the CRISPRO157:H7 qPCR assay for cattle feces spiked with pure cultures were 2.1 × 103 and 2.3 × 100 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples ( n = 576) was compared among the CRISPRO157:H7 qPCR assay, culture method, and four-plex qPCR assay. The CRISPRO157:H7 qPCR detected 42.2% of the samples (243 of 576 samples) as positive for E. coli O157:H7, compared with 30.4% (175 samples) by the culture method. Nearly all samples (97.2%; 560 samples) were positive for rfbEO157 by the four-plex PCR, but 21.8% (122 of 560 samples) were negative for the stx and/or eae genes, making it unlikely that EHEC O157:H7 was present in these samples. Cohen's kappa statistic indicated a fair and poor agreement beyond that due to chance between the CRISPR assay and the culture method and four-plex assay, respectively. This novel qPCR assay can detect the EHEC O157:H7 serotype in cattle feces by targeting CRISPR polymorphisms.

P048 NGS performance on a transplanted liver biopsy sample: A case study

A male pediatric patient was diagnosed with biliary atresia and was transplanted with liver from a deceased donor. The surgery went well, but years later the patient developed antibodies to Cw12, DR14, DQ7 and DQA1∗04:01 and various DP alleles. The need for high resolution typing to determine the identity of de novo DSA and the lack of any remaining pre-transplant sample on a deceased donor led to the use of NGS to determine chimeric typing. Genomic DNA was extracted from a post-transplant liver biopsy from this patient by using Gentra Puregene Tissue kit. We used the Immucor NGS FLEX HLA typing system for amplification and sequencing of HLA genes HLA -A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 followed by analysis with MIA FORA software. The NGS methodology is a clonal sequencing approach which solves the phase ambiguity problems inherent with Sanger-based sequencing when alleles are amplified and sequenced together. The genomic DNA extracted from the transplanted liver biopsy is chimeric, with residual recipient DNA at very low but detectable levels. The presence of chimeric reads is the result of co-amplification of both donor and recipient HLA genes. The majority of sequencing reads with zero mismatches belonged to the donor and were concordant with the deceased donor HLA type provided by UNOS. Unclaimed sequencing reads appeared as grey background and derived from the recipient HLA type (previously obtained with pre-transplant samples). In this case and a few other chimeric cases examined in our laboratory, the MIA FORA NGS software detected the HLA type of donor as the called alleles and showed some unclaimed reads that did not align to the donor type but rather to the HLA type of recipient. We note that if the donor and recipient HLA genes are perfectly matched in one or both alleles then they are indistinguishable. The clonal sequencing nature of the NGS reveals PCR primer specificity by identifying the sequences of all co-amplified genomic regions within the intended target region in the same biopsy sample. This case demonstrates the clinical utility of high resolution HLA typing by NGS technology in the clinical setting to de convolute a solid organ biopsy admixture i.e. the ability to do chimeric typing.

Comparative analysis of the EGFR, HER2, c-MYC, and MET variations in colorectal cancer determined by three different measures: gene copy number gain, amplification status and the 2013 ASCO/CAP guideline criterion for HER2 testing of breast cancer

BackgroundThe purpose of this study was to explore gene copy number (GCN) variation of EGFR, HER2, c-MYC, and MET in patients with primary colorectal cancer (CRC).MethodsDual-colour silver-enhanced in situ hybridization was performed in tissue samples of 334 primary CRC patients. The amplification status (GCN ratio ≥2) and GCN gain (average GCN ≥4) data for the EGFR, HER2, c-MYC and MET genes were obtained. GCN variation was also assessed by the criterion of the 2013 ASCO/CAP guidelines for HER2 testing.ResultsAmplification of EGFR, HER2, c-MYC and MET was detected in 8 (2.4%), 20 (6.0%), 29 (8.7%), and 14 (4.2%) patients, respectively. Of 66 patients with at least one amplified gene, five exhibited co-amplification of genes studied (HER2-MET co-amplification: two patients; HER2-c-MYC co-amplification: two patients; EGFR-c-MYC co-amplification: one patient). There were 109 patients with GCN gains of one or more genes (EGFR: 11/334, HER2: 29/334, c-MYC; 60/334, MET: 48/334) and 32.1% (35/109) had multiple GCN gains. When each GCN was assessed by the criterion of the ASCO/CAP 2013 guideline for HER2 testing, 116 people showed positive or equivocal results for one or more genes. The cumulative amplification status had no association with patients’ outcome. However, the cumulative results of the GCN gain and GCN status determined according to the ASCO/CAP guideline had a significant prognostic correlation in the univariate analysis (P values of 0.006 and 0.022, respectively). In the multivariate analysis, GCN gain and GCN status were independent prognostic factors (P values of 0.010 and 0.017, respectively).ConclusionsIn this study, we evaluated GCN variation of four genes in a large sample of Korean CRC patients. The amplification status was not related to patient outcome. However, the GCN gain and GCN status according to the ASCO/CAP 2013 guideline were independent prognostic factors.

Approaches to Controlled Co-Amplification of Genes for Production of Biopharmaceuticals: Study of the Insertion and Amplification Dynamics of Genetic Cassettes in the Genome of Chinese Hamster Ovary Cells during Co-Expression of Compatible Pair of Plasmids.

Plasmid vector family p1.1 based on non-coding regions of Chinese hamster housekeeping gene EEF1A and concatemer of Epstein-Barr virus terminal repeat increases the frequency of genome integration and provides rapid amplification of the target genes in the genome. For a pair of fluorescent proteins eGFP and mCherry it was shown that p1.1 vectors bearing dihydrofolate reductase and glutamine synthetase selection markers upon co-transfection into CHO DG44 cell line allow obtaining a polyclonal cell population in which ~70% of cells express both genes. The subsequent one-step gene amplification of the genome-integrated genetic cassettes under the selective pressure of increased concentrations of methotrexate can increase the expression of both integrated genes up to 8.2% eGFP and 9.9% mCherry of total protein. This approach can be used for the development of cell lines for the production of functional heterodimeric proteins, e.g. polypeptide hormones and therapeutic antibodies.

Selective DNA methylation in cancers controls collateral damage induced by large structural variations.

Chromosomal instability is a hallmark of human cancers, and is characterized by large structural variations in the genome. Such large structural variations are expected to create intrinsic collateral stress due to gene dosage changes in many genes that are co-deleted or co-amplified in large chromosomal segments (onco-passenger genes). We show that the tumor-toxic effects of gene dosage changes of onco-passenger genes are compensated by the uncoupling of their copy number variations from their expression by means of selective DNA methylation. For example, collateral co-amplification of genes in tumor suppressor pathways, such as the TGF-β and inflammatory signaling pathways, are compensated by DNA hypermethylation to suppress their overexpression, while collateral deletion of pro-oncogenic genes are compensated by DNA hypomethylation to promote their expression from the single remaining allele. Our work reveals an important tumorigenic mechanism of regulation of toxic gene copy number imbalance in tumor cells arising from chromosomal instability, and suggests that targeting the DNA methylation machinery may prevent compensatory regulation of onco-passenger gene expression in chromosomally unstable cancers, and re-activate dormant tumor suppressor pathways for effective therapy.

Prognostic impact of c-Rel nuclear expression and REL amplification and crosstalk between c-Rel and the p53 pathway in diffuse large B-cell lymphoma.

Dysregulated NF-κB signaling is critical for lymphomagenesis. The regulation, function, and clinical relevance of c-Rel/NF-κB activation in diffuse large B-cell lymphoma (DLBCL) have not been well studied. In this study we analyzed the prognostic significance and gene-expression signature of c-Rel nuclear expression as surrogate of c-Rel activation in 460 patients with de novo DLBCL. Nuclear c-Rel expression, observed in 137 (26.3%) DLBCL patients frequently associated with extranoal origin, did not show significantly prognostic impact in the overall- or germinal center B-like-DLBCL cohort, likely due to decreased pAKT and Myc levels, up-regulation of FOXP3, FOXO3, MEG3 and other tumor suppressors coincided with c-Rel nuclear expression, as well as the complicated relationships between NF-κB members and their overlapping function. However, c-Rel nuclear expression correlated with significantly poorer survival in p63+ and BCL-2- activated B-cell-like-DLBCL, and in DLBCL patients with TP53 mutations. Multivariate analysis indicated that after adjusting clinical parameters, c-Rel positivity was a significantly adverse prognostic factor in DLBCL patients with wild type TP53. Gene expression profiling suggested dysregulations of cell cycle, metabolism, adhesion, and migration associated with c-Rel activation. In contrast, REL amplification did not correlate with c-Rel nuclear expression and patient survival, likely due to co-amplification of genes that negatively regulate NF-κB activation. These insights into the expression, prognostic impact, regulation and function of c-Rel as well as its crosstalk with the p53 pathway underscore the importance of c-Rel and have significant therapeutic implications.