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Molecular cytogenetics, 11, 4
2018

Is cancer progression caused by gradual or simultaneous acquisitions of new chromosomes?

Bloomfield, Mathew, Duesberg, Peter

Foulds defined, "Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters" (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained "genetic instability" generates "variant sublines (with) changes in chromosome number" and that "clonal" progressions arise by "stepwise selection of more aggressive sublines". The literature, however, contains many examples of "immediate" selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates. In view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates "immediate" progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of "immediate" progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process. We conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the "late" clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression onfirming classical predictions based on DNA content

Digital object identifier (DOI): 10.1186/s13039-017-0350-4

Cancer letters, 412, 99--107
2018

Quantified postsurgical small cell size CTCs and EpCAM, [email protected], circulating tumor stem cells with cytogenetic abnormalities in hepatocellular carcinoma patients determine cancer relapse.

Wang, Liang, Li, Yilin, Xu, Jing, Zhang, Aiqun, Wang, Xuedong, Tang, Rui, Zhang, Xinjing, Yin, Hongfang, Liu, Manting, Wang, Daisy Dandan, Lin, Peter Ping, Shen, Lin, Dong, Jiahong

Detection of hepatocellular carcinoma circulating tumor cells performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such "uncapturable and invisible" CTCs. A novel SE-iFISH strategy, improved for detection of HCC CTCs in this study, was applied to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs (CD45 /CD31 ) in patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated. Existence of small cell size CTCs (≤5 μm of WBCs) with cytogenetic abnormality of aneuploid chromosome 8, which constituted majority of the detected CTCs in HCC patients, was demonstrated for the first time. The stemness marker EpCAM aneuploid circulating tumor stem cells (CTSCs), and EpCAM small CTCs with trisomy 8, promote tumor growth. Postsurgical quantity of small triploid CTCs (≥5 cells/6 ml blood), multiploid (≥pentasomy 8) CTSCs or CTM (either one ≥ 1) significantly correlated to HCC patients' poor prognosis, indicating that detection of those specific subtypes of CTCs and CTSCs in post-operative patients help predict neoplasm recurrence.

Digital object identifier (DOI): 10.1016/j.canlet.2017.10.004

PloS one, 13, e0190970
2018

Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma.

Hultman, Isabell, Haeggblom, Linnea, Rognmo, Ingvild, Jansson Edqvist, Josefin, Blomberg, Evelina, Ali, Rouknuddin, Phillips, Lottie, Sandstedt, Bengt, Kogner, Per, Shirazi Fard, Shahrzad, Ährlund-Richter, Lars

In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5-fold increase in Ki67-index (p<0.05), indicating that non-cycling (G0) cells entered the cell cycle following the doxorubicin exposure. Support for this notion was obtained also in vitro. A pharmacologically relevant dose (1μM) resulted in a marked accumulation of Ki67 positive BE(2)-C cells (p<0.0001), as well as a >3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growth-stimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation.

Digital object identifier (DOI): 10.1371/journal.pone.0190970

Investigative ophthalmology & visual science, 59, 561--571
2018

A Novel C-Terminal Mutation in Gsdma3 (C+/H-) Leads to Alopecia and Corneal Inflammatory Response in Mice.

Swirski, Sebastian, Röger, Carsten, Pienkowska-Schelling, Aldona, Ihlenburg, Cynthia, Fischer, Gösta, May, Oliver, Vorm, Mariann, Owczarek-Lipska, Marta, Neidhardt, John

Mutations in the gene encoding Gasdermin A3 (Gsdma3) have been described to cause severe skin phenotypes, including loss of sebaceous glands and alopecia, in mice. We discovered a novel C-terminal mutation in Gsdma3 in a new mouse line and characterized a less frequently reported corneal phenotype, likely caused by degeneration of Meibomian glands of the inner eyelid. We used histologic methods to evaluate the effects of the C+/H- mutation on sebaceous gland and skin morphology as well as Meibomian glands of the inner eyelid and corneal tissue. Chromosomal aberrations were excluded by karyogram analyses. The mutation was identified by Sanger sequencing of candidate genes. Analyses of skin samples from affected mice confirmed the frequently reported phenotypes associated with mutations in Gsdma3: Degeneration of sebaceous glands and complete loss of pelage. Immunologic staining of corneal samples suggested an inflammatory response with signs of neovascularization in half of the affected older mice. While the corneal phenotype was observed at irregular time points, mainly after 6 months, its appearance coincided with a degeneration of Meibomian glands in the eyelids of affected animals. The mutation described herein is associated with inflammation and neovascularization of corneal tissue. Simultaneous degeneration of Meibomian glands in affected animals suggested a change in tear-film composition as the underlying cause for the corneal phenotype. Our data further support that different pathogenic mechanisms underlie some of the reported mutations in Gsdma3.

Digital object identifier (DOI): 10.1167/iovs.17-22658

Pathology, research and practice, 214, 318--324
2018

Osteosarcoma arising in fibrous dysplasia, confirmed by mutational analysis of GNAS gene.

Sugiura, Yoshiya, Kanda, Hiroaki, Motoi, Noriko, Nomura, Kimie, Inamura, Kentaro, Okada, Erina, Matsumoto, Haruna, Shimoji, Takashi, Matsumoto, Seiichi, Nakayama, Jun, Takazawa, Yutaka, Ishikawa, Yuichi, Machinami, Rikuo

Malignancy arising in fibrous dysplasia (FD) is rare. Approximately 100 cases have been reported so far, and osteosarcoma is the most common malignancy. We report a case of osteosarcoma in a 33-year-old Japanese man with monostotic FD of the right proximal femur from the age of 16 years. Histologically, relatively well-differentiated osteosarcoma was found in the FD lesion. Immunohistochemically, the FD was negative for p53 or MDM2, and the MIB-1 index was less than 1%, whereas the osteosarcoma was positive for both p53 and MDM2, and the MIB-1 index was up to 15%. The FD and osteosarcoma were negative for CDK4. Fluorescent in situ hybridization assay showed no amplification of the MDM2 gene, indicating that the osteosarcoma was a conventional osteosarcoma, not an intraosseous well-differentiated type. The original cell of malignancy in FD is unclear. Malignancy can be potentially derived from dysplastic cells in the area of the FD or cells in the adjacent normal tissues. GNAS gene mutation has recently been reported for fibrous dysplasia and the mutation is highly specific to fibrous dysplasia among fibro-osseous lesions including osteosarcoma. In this case, point mutations of GNAS were found in the FD and osteosarcoma but not in the adjacent normal tissues, suggesting that osteosarcoma was derived from the spindle cells of FD. This is the first report to clearly show that osteosarcoma is derived from the spindle cells in fibrous dysplasia (FD).

Digital object identifier (DOI): 10.1016/j.prp.2017.10.018

Journal of Alzheimer's disease : JAD, 61, 899--905
2018

A Novel Antibody Targeting Tau Phosphorylated at Serine 235 Detects Neurofibrillary Tangles.

Brici, David, Götz, Jürgen, Nisbet, Rebecca M

Alzheimer's disease is characterized by two main pathological hallmarks in the human brain: the extracellular deposition of amyloid-β as plaques and the intracellular accumulation of the hyperphosphorylated protein tau as neurofibrillary tangles (NFTs). Phosphorylated tau (p-tau) specific-antibodies and silver staining have been used to reveal three morphological stages of NFT formation: pre-NFTs, intraneuronal NFTs (iNFTs), and extraneuronal NFTs (eNFTs). Here we characterize a novel monoclonal antibody, RN235, which is specific for tau phosphorylated at serine 235, and detects iNFTs and eNFTs in brain tissue, suggesting that phosphorylation at this site is indicative of late stage changes in tau.

Digital object identifier (DOI): 10.3233/JAD-170610

Nanoscale, 10, 4320--4331
2018

Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells.

Hausmann, Michael, Wagner, Emma, Lee, Jin-Ho, Schrock, Gerrit, Schaufler, Wladimir, Krufczik, Matthias, Papenfuß, Franziska, Port, Matthias, Bestvater, Felix, Scherthan, Harry

Ionizing radiation (IR)-induced damage confers functional and conformational changes to nuclear chromatin associated with DNA single and double strand breaks. This leads to the activation of complex DNA repair machineries that aim to preserve the integrity of the DNA molecule. Since hetero- and euchromatin are differentially accessible to DNA repair pathways, local chromatin re-arrangements and structural changes are among the consequences of an activated DNA damage response. Using super-resolution localization microscopy (SRLM), we investigated the X-ray-induced repositioning of γ-H2AX and histone H3K9me3 heterochromatin marks in the nuclei of HeLa cells. Aliquots of cells exposed to different IR doses (0.5, 1 and 2 Gy) were fixed at certain repair times for SRLM imaging. The number and size of nano-scale γ-H2AX molecule signal clusters detected increased with rising irradiation doses, with the number and size being the highest 0.5 h after irradiation. With growing repair time both the number and size of γ-H2AX nano-clusters decreased. Eight hours after irradiation, the number of clusters reached control levels, in agreement with the disappearance of most IR-induced foci seen by conventional microscopy. SRLM investigation of heterochromatin marks in spatial relation to γ-H2AX clusters showed that on average the heterochromatin density was high in the vicinity of γ-H2AX, which is in agreement with the observation that DSBs seem to relocate to the surface of heterochromatin clusters for DNA repair. The data demonstrate the potential of pointillist images obtained by SRLM for quantitative investigations of chromatin conformation changes and repair-protein recruitment on the nanoscale as measures for a radiation response.

Digital object identifier (DOI): 10.1039/c7nr08145f

Cancer genomics & proteomics, 15, 91--114
2018

Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5.

Kjeldsen, Eigil, Nielsen, Christine J F, Roy, Amit, Tesauro, Cinzia, Jakobsen, Ann-Katrine, Stougaard, Magnus, Knudsen, Birgitta R

Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations and a reduced modal chromosomal number compared to RPMI-8402. soaCGH analysis identified vast copy number alterations and >200 unbalanced DNA breakpoints distributed unevenly across the chromosomal complement in CPT-K5. In addition, the short tandem repeat alleles were found to be highly different between CPT-K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT.

Digital object identifier (DOI): 10.21873/cgp.20068

International journal of radiation biology, 94, 664--670
2018

Evaluation of chromosomal aberrations induced by , [email protected], Re-dendrimer nanosystem on B16f1 melanoma cells.

Tassano, Marcos, Oddone, Natalia, Fernández, Marcelo, Porcal, Williams, García, María Fernanda, Martínez-López, Wilner, Benech, Juan Claudio, Cabral, Pablo

To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with ReO . Biodistribution was performed administrating Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of Re-dendrimer in melanoma cells. Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15μCi (0.555 MBq) of Re-dendrimer for 24 h. Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.

Digital object identifier (DOI): 10.1080/09553002.2018.1478161

Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation
2018

Triploid Colubrid Snake Provides Insight into the Mechanism of Sex Determination in Advanced Snakes.

Rovatsos, Michail, Augstenová, Barbora, Altmanová, Marie, Sloboda, Michal, Kodym, Petr, Kratochvíl, Lukáš

The advanced snakes (Caenophidia), the important amniote lineage encompassing more than 3,000 living species, possess highly conserved female heterogamety across all families. However, we still lack any knowledge on the gene(s) and the molecular mechanism controlling sex determination. Triploid individuals spontaneously appear in populations of diploid species and can provide an important insight into the evolution of sex determination. Here, we report a case of spontaneous triploidy in a male of the twin-spotted ratsnake (Elaphe bimaculata) with ZZW sex chromosomes. We speculate that as both ZZ and ZZW individuals develop male gonads, the ratio between the number of Z chromosomes and autosomes, and not the presence of the W chromosome in the genome, drives sex determination in the advanced snakes.

Digital object identifier (DOI): 10.1159/000490124

Journal of clinical microbiology, 56
2018

Automated Interpretation of Blood Culture Gram Stains by Use of a Deep Convolutional Neural Network.

Smith, Kenneth P, Kang, Anthony D, Kirby, James E

Microscopic interpretation of stained smears is one of the most operator-dependent and time-intensive activities in the clinical microbiology laboratory. Here, we investigated application of an automated image acquisition and convolutional neural network (CNN)-based approach for automated Gram stain classification. Using an automated microscopy platform, uncoverslipped slides were scanned with a 40× dry objective, generating images of sufficient resolution for interpretation. We collected 25,488 images from positive blood culture Gram stains prepared during routine clinical workup. These images were used to generate 100,213 crops containing Gram-positive cocci in clusters, Gram-positive cocci in chains/pairs, Gram-negative rods, or background (no cells). These categories were targeted for proof-of-concept development as they are associated with the majority of bloodstream infections. Our CNN model achieved a classification accuracy of 94.9% on a test set of image crops. Receiver operating characteristic (ROC) curve analysis indicated a robust ability to differentiate between categories with an area under the curve of >0.98 for each. After training and validation, we applied the classification algorithm to new images collected from 189 whole slides without human intervention. Sensitivity and specificity were 98.4% and 75.0% for Gram-positive cocci in chains and pairs, 93.2% and 97.2% for Gram-positive cocci in clusters, and 96.3% and 98.1% for Gram-negative rods. Taken together, our data support a proof of concept for a fully automated classification methodology for blood-culture Gram stains. Importantly, the algorithm was highly adept at identifying image crops with organisms and could be used to present prescreened, classified crops to technologists to accelerate smear review. This concept could potentially be extended to all Gram stain interpretive activities in the clinical laboratory.

Digital object identifier (DOI): 10.1128/JCM.01521-17

Scientific reports, 8, 2286
2018

DNA damage in leukocytes after internal ex-vivo irradiation of blood with the α-emitter Ra-223.

Schumann, Sarah, Eberlein, Uta, Muhtadi, Razan, Lassmann, Michael, Scherthan, Harry

Irradiation with high linear energy transfer α-emitters, like the clinically used Ra-223 dichloride, severely damages cells and induces complex DNA damage including closely spaced double-strand breaks (DSBs). As the hematopoietic system is an organ-at-risk for the treatment, knowledge about Ra-223-induced DNA damage in blood leukocytes is highly desirable. Therefore, 36 blood samples from six healthy volunteers were exposed ex-vivo (in solution) to different concentrations of Ra-223. Absorbed doses to the blood were calculated assuming local energy deposition of all α- and β-particles of the decay, ranging from 0 to 142 mGy. γ-H2AX + 53BP1 co-staining and analysis was performed in leukocytes isolated from the irradiated blood samples. For DNA damage quantification, leukocyte samples were screened for occurrence of α-induced DNA damage tracks and small γ-H2AX + 53BP1 DSB foci. This revealed a linear relationship between the frequency of α-induced γ-H2AX damage tracks and the absorbed dose to the blood, while the frequency of small γ-H2AX + 53BP1 DSB foci indicative of β-irradiation was similar to baseline values, being in agreement with a negligible β-contribution (3.7%) to the total absorbed dose to the blood. Our calibration curve will contribute to the biodosimetry of Ra-223-treated patients and early after incorporation of α-emitters.

Digital object identifier (DOI): 10.1038/s41598-018-20364-7

Frontiers in neuroscience, 12, 55
2018

Safety and Efficacy of Scanning Ultrasound Treatment of Aged APP23 Mice.

Leinenga, Gerhard, Götz, Jürgen

Deposition of amyloid-β (Aβ) peptide leads to amyloid plaques that together with tau deposits characterize the brains of patients with Alzheimer's disease (AD). In modeling this pathology, transgenic animals such as the APP23 strain, that expresses a mutant form of the amyloid precursor protein found in familial cases of AD, have been instrumental. In previous studies, we have shown that repeated treatments with ultrasound in a scanning mode (termed scanning ultrasound or SUS) were effective in removing Aβ and restoring memory functions, without the need for a therapeutic agent such as an Aβ antibody. Considering that age is the most important risk factor for AD, we extended this study in which the mice were only 12 months old at the time of treatment by assessing a cohort of 2 year-old mice. Interestingly, at this age, APP23 mice are characterized by cerebral amyloid angiopathy (CAA) and the presence of occasional microbleeds. We found that SUS in aged mice that have been exposed to four SUS sessions that were spread out over 8 weeks and analyzed 4 weeks later did not show evidence of increased CAA or microbleeds. Furthermore, amyloid was reduced as assessed by methoxy-XO4 fluorescence. In addition, plaque-associated microglia were more numerous in SUS treated mice. Together this adds to the notion that SUS may be a treatment modality for human neurodegenerative diseases.

Digital object identifier (DOI): 10.3389/fnins.2018.00055

Postgraduate medical journal, 94, 398--403
2018

Adenotonsillar microbiome: an update.

Johnston, James Jordan, Douglas, Richard

Pathogenic bacteria associated with the adenoids and tonsils cause much morbidity in the paediatric population. Hyperplasia of the adenoids is associated with otitis media with effusion and hyperplasia of the palatine tonsils is associated with both recurrent tonsillitis and obstructive sleep apnoea. Most current knowledge of the microbiology of the upper airways has been derived from culture-based studies, which usually reflect only a small fraction of the bacteria present on the mucosal surface. Culture-independent molecular surveys based on 16S ribosomal RNA sequencing are now being employed to determine the microbiota on the surface and within the tissue of adenoids and palatine tonsils. This review describes the new techniques applied in determining the microbiome and summarises the results of studies employing these techniques.

Digital object identifier (DOI): 10.1136/postgradmedj-2018-135602

Evolution; international journal of organic evolution
2018

ZW, XY, and yet ZW: Sex chromosome evolution in snakes even more complicated.

Augstenová, Barbora, Johnson Pokorná, Martina, Altmanová, Marie, Frynta, Daniel, Rovatsos, Michail, Kratochvíl, Lukáš

Snakes are historically important in the formulation of several central concepts on the evolution of sex chromosomes. For over 50 years, it was believed that all snakes shared the same ZZ/ZW sex chromosomes, which are homomorphic and poorly differentiated in "basal" snakes such as pythons and boas, while heteromorphic and well differentiated in "advanced" (caenophidian) snakes. Recent molecular studies revealed that differentiated sex chromosomes are indeed shared among all families of caenophidian snakes, but that boas and pythons evolved likely independently male heterogamety (XX/XY sex chromosomes). The historical report of heteromorphic ZZ/ZW sex chromosomes in a boid snake was previously regarded as ambiguous. In the current study, we document heteromorphic ZZ/ZW sex chromosomes in a boid snake. A comparative approach suggests that these heteromorphic sex chromosomes evolved very recently and that they are poorly differentiated at the sequence level. Interestingly, two snake lineages with confirmed male heterogamety possess homomorphic sex chromosomes, but heteromorphic sex chromosomes are present in both snake lineages with female heterogamety. We point out that this phenomenon is more common across squamates. The presence of female heterogamety in non-caenophidian snakes indicates that the evolution of sex chromosomes in this lineage is much more complex than previously thought, making snakes an even better model system for the evolution of sex chromosomes.

Digital object identifier (DOI): 10.1111/evo.13543

Journal of Pathology Informatics, 9(1), 13
2018

Constant quest for quality: Digital cytopathology

Van Es, Simone L., Greaves, Janelle, Gay, Stephanie, Ross, Jennifer, Holzhauser, Derek, Badrick, Tony

Background: Special consideration should be given when creating and selecting cytopathology specimens for digitization to maximize quality. Advances in scanning and viewing technology can also improve whole-slide imaging (WSI) output quality. Methods: Accumulated laboratory experience with digitization of glass cytopathology slides was collected. Results: This paper describes characteristics of a cytopathology glass slide that can reduce quality on resulting WSI. Important points in the glass cytopathology slide selection process, preparation, scanning, and WSI-editing process that will maximize the quality of the resulting acquired digital image are covered. The paper outlines scanning solutions which have potential to predict issues with a glass cytopathology slide before image acquisition, allowing for adjustment of the scanning approach. WSI viewing solutions that better simulate the traditional microscope experience are also discussed. Conclusion: In addition to taking advantage of technical advances, practical steps can taken to maximize quality of cytopathology WSI.

Digital object identifier (DOI): 10.4103/jpi.jpi_6_18

Annals of diagnostic pathology, 34, 1--12
2018

Review with novel markers facilitates precise categorization of 41 cases of diagnostically challenging, "undifferentiated small round cell tumors". A clinicopathologic, immunophenotypic and molecular analysis.

Machado, Isidro, Yoshida, Akihiko, Morales, María Gema Nieto, Abrahão-Machado, Lucas Faria, Navarro, Samuel, Cruz, Julia, Lavernia, Javier, Parafioriti, Antonina, Picci, Piero, Llombart-Bosch, Antonio

Despite extensive immunohistochemical (IHC) and molecular studies combined with morphologic findings, a group of round/ovoid cell tumors histologically similar to Ewing sarcomas (ES) but lacking EWSR1-rearrangements may remain unclassifiable. We retrospectively analyzed 41 Ewing-like tumors (formalin-fixed, paraffin-embedded) previously determined as negative or non-informative for EWSR1-rearrangements by FISH and/or RT-PCR. A new histopathology revision and additional IHC and molecular analyses were carried out in order to investigate whether additional IHC and/or molecular testing in combination with the morphological findings may help in reaching a definitive diagnosis. Almost all the tumors (n=40) involved soft tissue and/or bone and half the patients died of disease. In the archival cases all diagnoses were Ewing sarcoma (ES), Ewing-like sarcoma (ELS), myoepithelial tumor and undifferentiated sarcoma (US). In the new review all the tumors were re-classified as, ES (n=16), Ewing-like tumor with EWSR1 rearrangement and amplification and possible EWSR1-NFATC2 gene fusion (n=1), CIC-rearranged sarcomas or undifferentiated sarcoma, most consistent with CIC-rearranged sarcoma (n=7), sarcoma with BCOR-alteration or undifferentiated sarcoma, consistent with BCOR-associated sarcoma (n=3), neuroblastoma (n=2), unclassifiable neoplasm with neuroblastic differentiation (n=1), malignant rhabdoid tumor (n=2), lymphoblastic lymphoma (n=1), clear cell sarcoma of the gastrointestinal tract (n=1), small cell carcinoma (n=1), sclerosing rhabdomyosarcoma (n=1), desmoplastic small round cell tumor (n=1), malignant peripheral sheath nerve tumor (n=1), poorly-differentiated synovial sarcoma (n=1), Possible gastrointestinal stromal tumor/GIST with predominant round cells (n=1) and possible SMARCA4-deficient-sarcoma (n=1). NKX2.2, ETV4 and BCOR immunoreactivity was observed in all ES, CIC-rearranged sarcomas and sarcomas with BCOR alteration, respectively. CIC-rearrangement by FISH was observed in many of the CIC-rearranged sarcomas. Our analysis of 41 Ewing-like tumors confirms that there may be a significant pathological and IHC overlap among Ewing-like tumors, with prognostic and therapeutic impacts. Additional IHC (NKX2.2, ETV4 and BCOR) and molecular studies including FUS, CIC or BCOR analysis may support the final diagnosis when FISH or RT-PCR fail to detect EWSR1-rearrangements. Any molecular findings should always be interpreted in relation to the specific clinical and pathological context.

Digital object identifier (DOI): 10.1016/j.anndiagpath.2017.11.011

British journal of cancer
2018

Heterogeneous MYCN amplification in neuroblastoma: a SIOP Europe Neuroblastoma Study.

Berbegall, Ana P, Bogen, Dominik, Pötschger, Ulrike, Beiske, Klaus, Bown, Nick, Combaret, Valérie, Defferrari, Raffaella, Jeison, Marta, Mazzocco, Katia, Varesio, Luigi, Vicha, Ales, Ash, Shifra, Castel, Victoria, Coze, Carole, Ladenstein, Ruth, Owens, Cormac, Papadakis, Vassilios, Ruud, Ellen, Amann, Gabriele, Sementa, Angela R, Navarro, Samuel, Ambros, Peter F, Noguera, Rosa, Ambros, Inge M

In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. Patients <18 months (18?m) showed a better outcome in all stages as compared to older patients (5-year OS in localised stages: <18?m: 0.95?±?0.04, >18?m: 0.67?±?0.14, p?=?0.011; metastatic: <18?m: 0.76?±?0.15, >18?m: 0.28?±?0.09, p?=?0.084). The genomic 'background', but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.

Digital object identifier (DOI): 10.1038/s41416-018-0098-6

The American journal of surgical pathology, 42, 656--664
2018

Clarifying the Distinction Between Malignant Peripheral Nerve Sheath Tumor and Dedifferentiated Liposarcoma: A Critical Reappraisal of the Diagnostic Utility of MDM2 and H3K27me3 Status.

Makise, Naohiro, Sekimizu, Masaya, Kubo, Takashi, Wakai, Susumu, Hiraoka, Nobuyoshi, Komiyama, Motokiyo, Fukayama, Masashi, Kawai, Akira, Ichikawa, Hitoshi, Yoshida, Akihiko

Malignant peripheral nerve sheath tumor (MPNST) and dedifferentiated liposarcoma (DDLPS) are 2 major types of pleomorphic spindle cell sarcoma. The differentiation of MPNST and DDLPS by histomorphology alone can be problematic. Although MDM2 amplification and PRC2 alteration leading to H3K27me3 deficiency are genetic hallmarks of DDLPS and MPNST, respectively, a small number of MDM2-amplified MPNSTs and H3K27me3-deficient DDLPSs have been reported in the literature. We systematically compared MDM2 and H3K27me3 status in 68 MPNSTs and 47 DDLPSs. Of the 62 MPNSTs, 22 were immunopositive for MDM2, mostly in a weak and/or focal manner. Of the 21 MDM2-positive MPNSTs successfully tested by fluorescence in situ hybridization, high-level MDM2 amplification was observed in 1 case. In contrast, MDM2 staining and high-level MDM2 amplification were positive in all the DDLPS tested (28/28 and 20/20). Of the 68 MPNSTs, 42 cases (62%) exhibited complete loss of H3K27me3. All the 13 MPNSTs that showed heterologous differentiation were deficient in H3K27me3. Of the 47 DDLPSs, 3 cases (6%) had complete loss of H3K27me3, all of which exhibited heterologous differentiation. One case of H3K27me3-deficient DDLPS exhibited homozygous loss of EED according to targeted next-generation sequencing, whereas there were no alterations in NF1 and CDKN2A. In conclusion, high-level MDM2 amplification strongly suggests DDLPS over MPNST. Although a good marker for MPNST, H3K27me3 deficiency also uncommonly occurs in DDLPS in association with PRC2 mutational inactivation. Because both markers are imperfectly specific, rare sarcomas with dual features could be encountered, and their classification should integrate other parameters.

Digital object identifier (DOI): 10.1097/PAS.0000000000001014

TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 131, 389--406
2018

Characterisation of Thinopyrum bessarabicum chromosomes through genome-wide introgressions into wheat.

Grewal, Surbhi, Yang, Caiyun, Edwards, Stella Hubbart, Scholefield, Duncan, Ashling, Stephen, Burridge, Amanda J, King, Ian P, King, Julie

Genome-wide introgressions of Thinopyrum bessarabicum into wheat resulted in 12 recombinant lines. Cytological and molecular techniques allowed mapping of 1150 SNP markers across all seven chromosomes of the J genome. Thinopyrum bessarabicum (2n = 2x = 14, JJ) is an important source for new genetic variation for wheat improvement due to its salinity tolerance and disease resistance. Its practical utilisation in wheat improvement can be facilitated through development of genome-wide introgressions leading to a variety of different wheat-Th . bessarabicum translocation lines. In this study, we report the generation of 12 such wheat-Th . bessarabicum recombinant lines, through two different crossing strategies, which were characterized using sequential single colour and multi-colour genomic in situ hybridization (sc-GISH and mc-GISH), multi-colour fluorescent in situ hybridization (mc-FISH) and single nucleotide polymorphic (SNP) DNA markers. We also detected 13 lines containing different Th. bessarabicum chromosome aberrations through sc-GISH. Through a combination of molecular and cytological analysis of all the 25 lines containing Th. bessarabicum recombinants and chromosome aberrations we were able to physically map 1150 SNP markers onto seven Th. bessarabicum J chromosomes which were divided into 36 segmental blocks. Comparative analysis of the physical map of Th. bessarabicum and the wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed that Th. bessarabicum contains the 4/5 translocation also present in the A genome of wheat. These wheat-Th . bessarabicum recombinant lines and SNP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Thinopyrum species containing the J or a closely-related genome such as Thinopyrum intermedium (J J J J StSt) and Thinopyrum elongatum (E E ), respectively.

Digital object identifier (DOI): 10.1007/s00122-017-3009-y