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Background: The HOXB13/IL17RB gene expression biomarker has been shown to predict response to adjuvant and extended endocrine therapy in patients with early-stage ER+ HER2- breast tumors. HOXB13 gene expression is the primary determinant driving the prognostic and endocrine treatment-predictive performance of the biomarker. Currently, there is limited data on HOXB13 expression in HER2+ and ER- breast cancers. Herein, we studied the expression of HOXB13 in large cohorts of HER2+ and ER- breast cancers. Methods: We investigated gene expression, genomic copy number, mutational signatures, and clinical outcome data in the TGGA and METABRIC breast cancer cohorts. Genomic-based gene amplification data was validated with tri-colored fluorescence in situ hybridization. Results: In the TCGA breast cancer cohort, HOXB13 gene expression was significantly higher in HER2+ versus HER2- breast cancers, and its expression was also significantly higher in the ER- versus ER+ breast cancers. HOXB13 is frequently co-gained or co-amplified with ERBB2. Joint copy gains of HOXB13 and ERBB2 occurred with low-level co-gains or high-level co-amplifications (co-amp), the latter of which is associated with an interstitial loss that includes the tumor suppressor BRCA1. ERBB2/HOXB13 co-amp tumors with interstitial BRCA1 loss exhibit a mutational signature associated with APOBEC deaminase activity and copy number signatures associated with chromothripsis and genomic instability. Among ERBB2-amplified tumors of different tissue origins, ERBB2/HOXB13 co-amp with a BRCA1 loss appeared to be enriched in breast cancer compared to other tumor types. Lastly, patients with ERBB2/HOXB13 co-amplified and BRCA1 lost tumors displayed a significantly shorter progression-free survival (PFS) than those with ERBB2-only amplifications. The difference in PFS was restricted to the ER- subset patients and this difference in PFS was not solely driven by HOXB13 gene expression. Conclusions: HOXB13 is frequently co-gained with ERBB2 at both low-copy number level or as complex high-level amplification with relative BRCA1 loss. ERBB2/HOXB13 amplified, BRCA1-lost tumors are strongly enriched in breast cancer, and patients with such breast tumors experience a shortened PFS. [ABSTRACT FROM AUTHOR]

Background: c-mesenchymal epithelial transition factor receptor (c-MET) and fibroblast growth factor receptor 2 (FGFR2) amplification have been identified as factors associated with advanced stage and poor prognosis in gastric cancer (GC). While they are typically considered mutually exclusive, concurrent amplifications have been reported in a small subset of GC patients. Methods: in this retrospective study, we analyzed the clinical outcomes of GC patients with MET and FGFR2 amplification using the next-generation sequencing (NGS) database cohort at Samsung Medical Center, which included a total of 2119 patients between October 2019 and April 2021. Results: Of 2119 cancer patients surveyed, the number of GC patients was 614 (29.0%). Out of 614 GC patients, 39 (6.4%) had FGFR2 amplification alone, 22 (3.6%) had MET amplification, and 2 GC patients (0.3%) had concurrent FGFR2 and MET amplification. Two patients with concurrent FGFR2 and MET amplification did not respond to first-line chemotherapy. These two patients had significantly shorter overall survival (3.6 months) compared to patients with FGFR2 or MET amplification alone (13.6 months and 8.4 months, respectively) (p = 0.004). Lastly, we tested the existence of FGFR2 and MET in tumor specimens from different organ sites. Initially, the NGS was tested in a primary tumor specimen from stomach cancer, where the MET copy number was 14.1 and the FGFR2 copy number was 5.3. We confirmed that both MET and FGFR2 were highly amplified in the primary tumor using FISH (MET–CEP7 ratio = 5 and FGFR2–CEP7 ratio = 3). However, although the MET copy number was normal in peritoneal seeding using FISH, FGFR2 remained amplified using FISH (FGFR2–CEP7 ratio = 7) with high FGFR2 protein overexpression. Hence, there was intra-patient molecular heterogeneity. Conclusions: our findings suggest that concurrent amplification of FGFR2 and MET in GC patients is associated with clinical aggressiveness and may contribute to non-responsiveness to chemotherapy or targeted therapy. [ABSTRACT FROM AUTHOR]

The locus at 17q12 erb‐b2 receptor tyrosine kinase 2 (ERBB2) has been heavily amplificated and overexpressed in gastric cancer (GC), but it remains to be elucidated about the clinical significance of the co‐amplification and co‐overexpression of PGAP3 gene located around ERBB2 in GC. The profile of PGAP3 and ERBB2 in four GC cell lines and tissue microarrays containing 418 primary GC tissues was assessed to investigate the co‐overexpression and clinical significance of the co‐amplified genes, and to evaluate the impact of the co‐amplified genes on the malignancy of GC. Co‐amplification of PGAP3 and ERBB2 accompanied with co‐overexpression was observed in a haploid chromosome 17 of NCI‐N87 cells with double minutes (DMs). PGAP3 and ERBB2 were overexpressed and positively correlated in 418 GC patients. Co‐overexpression of the PGAP3 and ERBB2 was correlated with T stage, TNM stage, tumour size, intestinal histological type and poor survival proportion in 141 GC patients. In vitro, knockdown of the endogenous PGAP3 or ERBB2 decreased cell proliferation and invasion, increased G1 phase accumulation and induced apoptosis in NCI‐N87 cells. Furthermore, combined silencing of PGAP3 and ERBB2 showed an additive effect on resisting proliferation of NCI‐N87 cells compared with targeting ERBB2 or PGAP3 alone. Taken together, the co‐overexpression of PGAP3 and ERBB2 may be crucial due to its significant correlation with clinicopathological factors of GC. Haploid gain of PGAP3 co‐amplified with ERBB2 is sufficient to facilitate the malignancy and progression of GC cells in a synergistic way. [ABSTRACT FROM AUTHOR]

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80 ng/0.1 ng) or a mix of the menstrual blood/semen DNA (5 ng/0.1 ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1 ng semen DNA, even in the presence of 5 ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases. [ABSTRACT FROM AUTHOR]

The aim of the present study was to identify potential human epidermal growth factor receptor 2 (HER2) amplification, according to American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) 2013 HER2 testing guidelines, in patients previously determined not to possess HER2 amplification, in accordance with previous 2007 guidelines. Potential discrepancies may arise from chromosome enumeration probe 17 (CEP17) amplification, deletion, polysomyor monosomy. HER2, CEP17, tumor protein p53 (TP53) and retinoic acid receptor α (RARA) genes from 67 patient specimens with suspected amplification, polysomy or monosomy of CEP17 were analyzed using fluorescence in situ hybridization. HER2 status was interpreted using 2007 and 2013 ASCO HER2 test guidelines as well as the reference genes TP53 and RARA. According to ASCO/CAP2007 HER2 guidelines, 20 patients exhibited HER2 amplification (29.85%), 41 were without HER2 amplification (including 25 with polysomy, 15 with monosomy and 1 with suspected monosomy plus co-amplification of HER2 and CEP17) and the remaining 6 patients were equivocal. Using ASCO/CAP 2013 HER2 g uidelines, 4 9 patients e xhibited HER2 gene a mplification (73.1%). T he 29-patient i ncrease included 6 originally at equivocal levels but now demonstrating amplification, 22 originally with polysomy but now revealing co-amplification, and 1 with suspected monosomy plus co-amplification of HER2 and CEP17. According to TP53 and RARA, HER2 was amplified in 43 patients (64.1%). Using the revised guidelines, HER2, originally identified as amplified in 6 patients, was not amplified following the introduction of TP53 and RARA control genes. Among these 6, 4 possessed normal TP53 and RARA. The incidence of co-amplification of HER2 and CEP17 was 1.4% (21/1,518). RARA and TP53 are suitable control genes to evaluate HER2 status. [ABSTRACT FROM AUTHOR]

Background Bovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions. Methods During the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard. Results Three of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed. Conclusions The present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation. [ABSTRACT FROM AUTHOR]

Co-amplification of the centromere on chromosome 17 ( CEP17) and HER2 can occur in breast cancer. Such aberrant patterns (clusters) on CEP17 can be misleading to calculate the HER2/CEP17 ratio, and thus underreporting of HER2 amplification. We identified 14 breast cancers retrospectively with HER2/CEP17 co-amplification and performed FISH (fluorescence in situ hybridization) with additional chromosome 17 probes (17p11.1-q11.1, 17p11.2-p12, TP 53 on 17p13.1, RARA on 17q21.1-3 and TOP2 on 17q21.3-22) to characterize the spanning of the amplicon in these cases. Furthermore, the HER2 status was analyzed by means of HER2 silver in situ hybridization (SISH) and immunohistochemistry (IHC). The co-amplification of HER2/ CEP17 was compared between the three institutions. TP53 was eusomic in all cases, 17p11.2-p12 in 79% (11/14), whereas 17p11.1-q11.1 showed chromosomal gain in all cases. RARA was amplified in 10/14 cases (71%) and TOP2 in 3/14 cases (21%). HER2 was amplified with FISH/SISH in all 14 cases. 9/14 tumors were 3+ IHC positive (64%) and 3 cases were 2+ IHC positive. In our cohort the CEP17 amplicon almost always involves the HER2 but not the TOP2 locus. Overall agreement on HER2/CEP17 ratio (when applying ASCO/CAP guidelines) was only 64% (9/14 cases) between the institutions. Discrepant ratios varied from 1.1 to 14.3. The HER2/CEP17 co-amplification is not defined in the ASCO/CAP guidelines, and may result in inaccurate HER2-FISH/SISH status, particularly if only the calculated HER2/CEP17 ratio is reported. It is recommended to report separate CEP17 and HER2 signals in complex HER2/CEP17 patterns. [ABSTRACT FROM AUTHOR]

A cell suspension culture was established from a transgenic petunia ( Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II ( nptII) and β-glucuronidase ( uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 μg/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 μg/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement. [ABSTRACT FROM AUTHOR]

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Background: Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus isolate classification) and target groups. Results: We performed an intensive literature research, reshuffled existing primers into new pairs, designed new Illumina-primers, and annealing blocking oligonucleotides. A final number of 439 primer pairs were subjected to in silico PCRs. Best primer pairs were selected and experimentally tested. The most promising primer pair with a small amplicon size, nu-SSU-1333-5′/nu-SSU-1647-3′ (FF390/FR-1), was successful in describing fungal communities by Illumina sequencing. Results were confirmed by a simultaneous metagenomics and eukaryote-specific primer approach. Co-amplification occurred in all sample types but was effectively reduced by blocking oligonucleotides. Conclusions: The compiled data revealed the presence of an enormous diversity of fungal 18S rRNA gene primer pairs in terms of fungal coverage, phylum spectrum and co-amplification. Therefore, the primer pair has to be carefully selected to fulfill the requirements of the individual research projects. The presented primer toolkit offers comprehensive lists of 164 primers, 439 primer combinations, 4 blocking oligonucleotides, and top primer pairs holding all relevant information including primer's characteristics and performance to facilitate primer pair selection. [ABSTRACT FROM AUTHOR]