Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™–HER2-FISH workflow

Background In breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology. Methods 50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany). Results Applying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive. Conclusions The DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

Deciphering the Mechanism of Glyphosate Resistance in Amaranthus palmeri by Cytogenomics

In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in <i>Amaranthus palmeri</i> (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which <i>5-enolpyruvylshikimate-3-phosphate synthase</i> (<i>EPSPS</i>) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to &#x3e;160-fold increase in copies of the <i>EPSPS</i> gene than in a glyphosate-susceptible (GS) population. This increased copy number of the <i>EPSPS</i> gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb <i>EPSPS</i> cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified <i>EPSPS</i> copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The <i>EPSPS</i> gene-containing eccDNA having a size of ∼400 kb is termed <i>EPSPS</i>-eccDNA and showed somatic mosacism in size and copy number. <i>EPSPS</i>-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the <i>EPSPS</i> locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of <i>EPSPS</i>-eccDNA sheds light on various characteristics of <i>EPSPS</i>-eccDNA that favor GR in AP.

An Integrated Epigenomic and Genomic View on Phyllodes and Phyllodes-Like Breast Tumors

Fibroepithelial lesions (FL) of the breast, in particular Phyllodes tumors (PT) and fibroadenomas, pose a significant diagnostic challenge. There are no generally accepted criteria that distinguish benign, borderline, malignant PT, and FA. Combined genome-wide DNA methylation and copy number variant (CNV) profiling is an emerging strategy to classify tumors. We compiled a series of patient-derived archival biopsy specimens reflecting the FL spectrum and histological mimickers including clinical follow-up data. DNA methylation and CNVs were determined by well-established microarrays. Comparison of the patterns with a pan-cancer dataset assembled from public resources including "The Cancer Genome Atlas" (TCGA) and "Gene Expression Omnibus" (GEO) suggests that FLs form a methylation class distinct from both control breast tissue as well as common breast cancers. Complex CNVs were enriched in clinically aggressive FLs. Subsequent fluorescence in situ hybridization (FISH) analysis detected respective aberrations in the neoplastic mesenchymal component of FLs only, confirming that the epithelial component is non-neoplastic. Of note, our approach could lead to the elimination of the diagnostically problematic category of borderline PT and allow for optimized prognostic patient stratification. Furthermore, the identified recurrent genomic aberrations such as 1q gains (including MDM4), CDKN2a/b deletions and EGFR amplifications may inform therapeutic decision-making.

Primary Sclerosing Epithelioid Fibrosarcoma of the Kidney: A Case Report and Review of the Literature

Sclerosing epithelioid fibrosarcoma (SEF) is a rare variant of fibrosarcoma. We report one case of primary kidney SEF occurring in a 38-year-old man. Microscopically, epithelioid neoplastic cells are mainly arranged in cords and nests embedded in the dense sclerosing stroma. Diffuse immunohistochemical staining for MUC4 in neoplastic cells and the presence of the EWSR1 gene split by fluorescence in situ hybridization (FISH) analysis confirmed the histological diagnosis. Primary kidney SEF is extremely rare, the differential diagnosis strategy broadly includes a series of tumors with epithelioid morphology and sclerosing matrix, mainly including sclerosing variants of clear cell sarcoma of the kidney (CCSK), renal synovial sarcoma (SS), renal solitary fibrous tumor (SFT), metanephric stromal tumor (MST), sclerosing perivascular epithelioid cell tumor (PEComa), and carcinomas, and immunohistochemical expression of MUC4 and evidence of the EWSR1 gene split are helpful in making a definite diagnosis.

Low-Molecular-Weight Seaweed-Derived Polysaccharides Lead to Increased Faecal Bulk but Do Not Alter Human Gut Health Markers

Seaweeds are potentially sustainable crops and are receiving significant interest because of their rich bioactive compound content; including fatty acids, polyphenols, carotenoids, and complex polysaccharides. However, there is little information on the in vivo effects on gut health of the polysaccharides and their low-molecular-weight derivatives. Herein, we describe the first investigation into the prebiotic potential of low-molecular-weight polysaccharides (LMWPs) derived from alginate and agar in order to validate their in vivo efficacy. We conducted a randomized; placebo-controlled trial testing the impact of alginate and agar LWMPs on faecal weight and other markers of gut health and on composition of gut microbiota. We show that these LMWPs led to significantly increased faecal bulk (20–30%). Analysis of gut microbiome composition by sequencing indicated no significant changes attributable to treatment at the phylum and family level, although FISH analysis showed an increase in Faecalibacterium prausnitzii in subjects consuming agar LMWP. Sequence analysis of gut bacteria corroborated with the FISH data, indicating that alginate and agar LWMPs do not alter human gut microbiome health markers. Crucially, our findings suggest an urgent need for robust and rigorous human in vivo testing—in particular, using refined seaweed extracts.

Transcriptome Analysis Reveals Sexual Disparities between Olfactory and Immune Gene Expression in the Olfactory Epithelium of Megalobrama amblycephala

The olfactory organ is an important chemoreceptor in vertebrates. However, the sexual disparities in gene expression patterns in the olfactory organ in fish remain unstudied. Here, we conducted a transcriptome analysis of the olfactory epithelium (OE) of male and female blunt snout bream (Megalobrama amblycephala) to identify the differences. The histological analysis showed that there were 22 leaf-like olfactory lamellaes on one side of the OE of the adult blunt snout bream. The sensory area of OE is enriched with ciliated receptor cells and microvilli receptor cells. The transcriptome analysis showed that only 10 out of 336 olfactory receptor genes (224 ORs, 5 V1Rs, 55 V2Rs, and 52 TAARs) exhibited significant expression differences between males and females, and most of the differentially expressed genes were related to the immune system. We also validated these results using qPCR: 10 OR genes and 6 immunity-related genes significantly differed between males and females. The FISH analysis results indicated that the ORs were mainly expressed at the edge of the olfactory lamellae. Collectively, our study reveals that gender is not an important factor influencing the expression of olfactory receptors, but the expression of immune genes varies greatly between the genders in blunt snout bream.

Mad2 Induced Aneuploidy Contributes to Eml4-Alk Driven Lung Cancer by Generating an Immunosuppressive Environment

Aneuploidy, an imbalance number of chromosomes, is frequently observed in lung cancer and inversely correlates with patient survival. Paradoxically, an aneuploid karyotype has detrimental consequences on cellular fitness, and it has been proposed that aneuploid cells, at least in vitro, generate signals for their own elimination by NK cells. However, how aneuploidy affects tumor progression as well as the interplay between aneuploid tumor cells and the tumor microenvironment is still unclear. We generated a new mouse model in which overexpression of Mad2 was almost entirely restricted to normal epithelial cells of the lung, and combined it with an oncogenic Eml4-Alk chromosome inversion. This combination resulted in a higher tumor burden and an increased number of tumor nodules compared to control Eml4-Alk mice alone. The FISH analysis detected significant differences in the aneuploidy levels in the non-tumor regions of Eml4-Alk+Mad2 compared to Eml4-Alk alone, although both tumor groups presented similar levels of aneuploidy. We further show that aneuploid cells in the non-tumor areas adjacent to lung tumors recruit immune cells, such as tumor-associated macrophages. In fact, these areas presented an increase in alveolar macrophages, neutrophils, decreased cytotoxic CD8+ T cells, and IFN-γ, suggesting that aneuploid cells in the surrounding tumor areas create an immunosuppressive signature that might contribute to lung tumor initiation and progression.

An Infertile Azoospermic Male with 45, X T(Yp;15) Karyotype

Objective: 45, X is a very rare condition that usually results from Y/autosomal translocations or insertions. Here we present an infertile azoospermic man who had 45, X t(Yp;15) karyotype and deletion of AZF (azoospermia factor) gene region. Case report: A 35-year-old infertile azoospermic man with a typical male appearance came for infertility genetic counseling. He was infertile for more than ten years and had short height. High-resolution of metaphase chromosomes of 50 peripheral white blood cells were analyzed for karyotyping. Fluorescence in situ hybridization (FISH) analysis and Polymerase chain reaction (PCR) were done for SRY and AZF gene localization. Karyotyping and FISH analysis revealed 45, X t(Yp;15) karyotype and no mosaicism. More investigation on the Y chromosome revealed no deletion in the SRY region, but AZF a/b/c were deleted. It was revealed that Yp's subtelomeric region but not Yq was translocated to chromosome 15. Conclusion: This study shows that despite the lack of a complete Y chromosome in this person, the occurrence of secondary male traits is a result of the short arm translocation of the Y chromosome, which contains the (ex-determining region Y) SRY gene. Infertility is also due to the Y chromosomes long arm's deletion containing the AZF gene region.

Fine Breakpoint Mapping by Genome Sequencing Reveals the First Large X Inversion Disrupting the NHS Gene in a Patient with Syndromic Cataracts

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.