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Diagnostics (Basel, Switzerland), 8
2018

Aneuploid CTC and CEC.

Lin, Peter Ping

Conventional circulating tumor cell (CTC) detection technologies are restricted to large tumor cells (> white blood cells (WBCs)), or those unique carcinoma cells with double positive expression of surface epithelial cell adhesion molecule (EpCAM) for isolation, and intracellular structural protein cytokeratins (CKs) for identification. With respect to detecting the full spectrum of highly heterogeneous circulating rare cells (CRCs), including CTCs and circulating endothelial cells (CECs), it is imperative to develop a strategy systematically coordinating all tri-elements of nucleic acids, biomarker proteins, and cellular morphology, to effectively enrich and comprehensively identify CRCs. Accordingly, a novel strategy integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), independent of cell size variation and free of hypotonic damage as well as anti-EpCAM perturbing, has been demonstrated to enable in situ phenotyping multi-protein expression, karyotyping chromosome aneuploidy, and detecting cytogenetic rearrangements of the gene in non-hematologic CRCs. Symbolic non-synonymous single nucleotide variants (SNVs) of both the gene (P33R) in each single aneuploid CTCs, and the cyclin-dependent kinase inhibitor 2A ( ) tumor suppressor gene in each examined aneuploid CECs, were identified for the first time across patients with diverse carcinomas. Comprehensive co-detecting observable aneuploid CTCs and CECs by SE-iFISH, along with applicable genomic and/or proteomic single cell molecular profiling, are anticipated to facilitate elucidating how those disparate categories of aneuploid CTCs and CECs cross-talk and functionally interplay with tumor angiogenesis, therapeutic drug resistance, tumor progression, and cancer metastasis.

Digital object identifier (DOI): 10.3390/diagnostics8020026

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

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

Surgical oncology, 27, 106--113
2018

Detection of RET (rearranged during transfection) variants and their downstream signal molecules in RET rearranged lung adenocarcinoma patients.

Kim, Jeong-Oh, Shin, Jung-Young, Kim, Min Young, Son, Kyoung Hwa, Jung, Chan Kwon, Kim, Tae-Jung, Kim, Su Young, Park, Jae Kil, Sung, Sook Whan, Bae, Sang Ju, Min, Hyun Jung, Kang, Jin-Hyoung

We screened resected tumor tissues from patients with lung cancer for EGFR mutations, ALK rearrangements, and rearranged during transfection (RET) gene variants (including RET rearrangements and the Kinesin Family Member 5B (KIF5B)-RET fusion gene) using various methods including reverse transcription polymerase chain reaction (RT-PCR), transcript assays, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). We also examined the protein expression of associated downstream signaling molecules to assess the effect of these variants on patient outcome. We constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from patients with lung adenocarcinoma and analyzed the microarray by both FISH (using RET break-apart and KIF5B-RET SY translocation probes) and a commercial RET transcript assay. We evaluated the expression of RET and RET-related signaling molecules, including p-AKT and p-ERK, by TMA -based IHC staining. Among the 581 specimens, 51 (8.8%) specimens harbored RET rearrangements, including 12 cases (2.1%) carrying a KIF5B-RET fusion gene. Surprisingly, RET expression was lower in KIF5B-RET fusion gene-positive than in RET wild-type specimens. We detected activating EGFR mutations in 11 (21.6%) of the 51 RET variant-positive specimens. Among the KIF5B-RET fusion gene-positive specimens, p-ERK expression was significantly lower in the EGFR mutation subgroup showing RET expression than in the EGFR mutation subgroup that did not express RET. Similarly, the RET rearrangement group showed significant variation in the expression level of p-AKT (P?=?0.028) and p-ERK, whose expression remarkably increased in specimens not expressing RET. The expression of p-ERK markedly increased in the RET rearrangement group regardless of RET expression. This result suggests that a combination of RET and ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring RET variants.

Digital object identifier (DOI): 10.1016/j.suronc.2018.01.006

Scientific Reports, 8(1), 1141
2018

First experimental proof of Proton Boron Capture Therapy (PBCT) to enhance protontherapy effectiveness

Cirrone, GAP, Manti, L, Margarone, D, Petringa, G, Giuffrida, L, Minopoli, A, Picciotto, A, Russo, G, Cammarata, F, Pisciotta, P, others

Protontherapy is hadrontherapy’s fastest-growing modality and a pillar in the battle against cancer. Hadrontherapy’s superiority lies in its inverted depth-dose profile, hence tumour-confined irradiation. Protons, however, lack distinct radiobiological advantages over photons or electrons. Higher LET (Linear Energy Transfer) 12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in efficient cell killing, i.e. higher Relative Biological Effectiveness (RBE). However, economic and radiobiological issues hamper 12C-ion clinical amenability. Thus, enhancing proton RBE is desirable. To this end, we exploited the p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam. To maximize the reaction rate, we used sodium borocaptate (BSH) with natural boron content. Boron-Neutron Capture Therapy (BNCT) uses 10B-enriched BSH for neutron irradiation-triggered alpha particles. We recorded significantly increased cellular lethality and chromosome aberration complexity. A strategy combining protontherapy’s ballistic precision with the higher RBE promised by BNCT and 12C-ion therapy is thus demonstrated.

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

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

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

Journal of medical entomology, 55, 575--586
2018

Description of Larval Instars To Fill a Gap in Forensic Entomology: The Larvae of Paralucilia pseudolyrcea (Diptera: Calliphoridae).

Da Silva, S M, Vairo, K P, Moura, M O

A fundamental assumption of forensic entomology for estimating the postmortem interval is that insect species are accurately identified, which depends on diagnostic morphological characters. Larvae of the blow fly Paralucilia pseudolyrcea (Mello, 1969) (Diptera: Calliphoridae) were sampled from four corpses in the state of Paraná, Brazil, but despite the forensic importance of this species, morphological data for the identification of its larval instars are lacking, limiting its usefulness in such cases. Thus, the main goal of this study was to describe the larval instars of P. pseudolyrcea. The material was obtained from a colony established by larvae collected from a corpse of a murder case. Overall, the distribution of spines is a key character for identifying this species in the first, second and third instars. Other characteristics, such as the presence of an accessory oral sclerite, the small cirri, the number of lobes of the anterior spiracle and the morphology of posterior spiracles, separates P. pseudolyrcea from other necrophagous blow flies. The detailed morphological description provided here facilitates the identification of larval instars of P. pseudolyrcea and their differentiation from those of other calliphorid species.

Digital object identifier (DOI): 10.1093/jme/tjx257

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

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

Environmental science and pollution research international
2018

Toxicological evaluation of nail polish waste discarded in the environment.

Felzenszwalb, Israel, Fernandes, Andreia da Silva, Brito, Lara Barroso, Oliveira, Gisele Augusto Rodrigues, Silva, Paula Aquino Soeiro, Arcanjo, Maria Elena, Marques, Monica Regina da Costa, Vicari, Taynah, Leme, Daniela Morais, Cestari, Marta Margarete, Ferraz, Elisa Raquel Anastacio

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Digital object identifier (DOI): 10.1007/s11356-018-1880-y

Radiology, 288, 529--535
2018

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

Suntharalingam, Saravanabavaan, Mladenov, Emil, Sarabhai, Theresia, Wetter, Axel, Kraff, Oliver, Quick, Harald H, Forsting, Michael, Iliakis, Georg, Nassenstein, Kai

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, ?-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes.

Digital object identifier (DOI): 10.1148/radiol.2018172453

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

PloS one, 13, e0193213
2018

Impact of 9p deletion and p16, Cyclin D1, and Myc hyperexpression on the outcome of anaplastic oligodendrogliomas.

Michaud, Karine, de Tayrac, Marie, D'Astous, Myreille, Paquet, Claudie, Gould, Peter Vincent, Saikali, Stéphan

To study the presence of 9p deletion and p16, cyclin D1 and Myc expression and their respective diagnostic and prognostic interest in oligodendrogliomas. We analyzed a retrospective series of 40 consecutive anaplastic oligodendrogliomas (OIII) from a single institution and compared them to a control series of 10 low grade oligodendrogliomas (OII). Automated FISH analysis of chromosome 9p status and immunohistochemistry for p16, cyclin D1 and Myc was performed for all cases and correlated with clinical and histological data, event free survival (EFS) and overall survival (OS). Chromosome 9p deletion was observed in 55% of OIII (22/40) but not in OII. Deletion was highly correlated to EFS (median = 29 versus 53 months, p<0.0001) and OS (median = 48 versus 83 months, p<0.0001) in both the total cohort and the OIII population. In 9p non-deleted oligodendrogliomas, p16 hyperexpression correlated with a shorter OS (p = 0.02 in OII and p = 0.0001 in OIII) whereas lack of p16 expression was correlated to a shorter EFS and OS in 9p deleted OIII (p = 0.001 and p = 0.0002 respectively). Expression of Cyclin D1 was significantly higher in OIII (median expression 45% versus 14% for OII, p = 0.0006) and was correlated with MIB-1 expression (p<0.0001), vascular proliferation (p = 0.002), tumor necrosis (p = 0.04) and a shorter EFS in the total cohort (p = 0.05). Hyperexpression of Myc was correlated to grade (median expression 27% in OII versus 35% in OIII, p = 0.03), and to a shorter EFS in 9p non-deleted OIII (p = 0.01). Chromosome 9p deletion identifies a subset of OIII with significantly worse prognosis. The combination of 9p status and p16 expression level identifies two distinct OIII populations with divergent prognosis. Hyperexpression of Bcl1 and Myc appears highly linked to anaplasia but the prognostic value is unclear and should be investigated further.

Digital object identifier (DOI): 10.1371/journal.pone.0193213

Journal of clinical pathology
2018

KRAS fluorescence in situ hybridisation testing for the detection and diagnosis of pancreatic adenocarcinoma.

Shiroma, Noriyuki, Arihiro, Koji, Oda, Miyo, Orita, Makoto

The aim of our study was to analyse correlations between mutation status, chromosomal changes that affect status in cells from pancreatic tumours. We collected 69 cases of surgically resected pancreatic ductal adenocarcinoma (PDA) and seven cases of chronic pancreatitis (CP). Chromosomal abnormalities of and CEP12 were detected using fluorescence in situ hybridisation (FISH). The number of CEP12 signals per cell ranged from 1.78 to 2.04 and 1.46 to 4.88 in CP and PDA samples, respectively, while the number of signals per cell ranged from 1.94 to 2.06 and 1.88 to 8.18 in CP and PDA samples, respectively. The 'chromosomal instability index', which was defined as the percentage of cells with any chromosomal abnormality, was over 5.7 times greater in PDA than in CP. We performed mutation analysis by direct sequencing and found that tumours with mutations have a significantly higher mean signal per cell from PDA samples compared with tumours with wild-type amplification was noted in 10% of cases. Although we found that lymph node metastasis and distal metastasis of PDA were more frequent in cases with amplification, this was not correlated with overall survival. Using a threshold of 40%, we found that the chromosomal instability index robustly discriminated PDA cells from CP cells. Based on these findings, we concluded that FISH testing of using cytology samples may represent an accurate approach for the diagnosis of PDA.

Digital object identifier (DOI): 10.1136/jclinpath-2018-205002

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

Biological procedures online, 20, 13
2018

Optimization of Immunofluorescent Detection of Bone Marrow Disseminated Tumor Cells.

Axelrod, Haley D, Pienta, Kenneth J, Valkenburg, Kenneth C

Cancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal. We have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow. Robust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis.

Digital object identifier (DOI): 10.1186/s12575-018-0078-5

Cancers, 10
2018

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes.

M'kacher, Radhia, Cuceu, Corina, Al Jawhari, Mustafa, Morat, Luc, Frenzel, Monika, Shim, Grace, Lenain, Aude, Hempel, William M, Junker, Steffen, Girinsky, Theodore, Colicchio, Bruno, Dieterlen, Alain, Heidingsfelder, Leonhard, Borie, Claire, Oudrhiri, Noufissa, Bennaceur-Griscelli, Annelise, Moralès, Olivier, Renaud, Sarah, Van de Wyngaert, Zoé, Jeandidier, Eric, Delhem, Nadira, Carde, Patrice

: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. : Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. : Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30-/CD15- cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival ( < 10 ), event-free survival ( < 10 ), and freedom from progression ( < 10 ) and the presence of an ALT profile in lymph nodes of EBV+ patients. : The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.

Digital object identifier (DOI): 10.3390/cancers10060169