scholarly journals Recurring Translocations in Barrett’s Esophageal Adenocarcinoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Manisha Bajpai ◽  
Anshuman Panda ◽  
Kristen Birudaraju ◽  
James Van Gurp ◽  
Amitabh Chak ◽  
...  
Keyword(s):  
Esophageal Adenocarcinoma ◽  
Chromosomal Translocation ◽  
Diagnostic Assays ◽  
Specificity And Sensitivity ◽  
Chromosomal Breakpoints ◽  
Chromosomal Changes ◽  
Microscopy Techniques ◽  
Formalin Fixed

Barrett’s esophagus (BE) is a premalignant metaplasia in patients with chronic gastroesophageal reflux disease (GERD). BE can progress to esophageal adenocarcinoma (EA) with less than 15% 5-year survival. Chromosomal aneuploidy, deletions, and duplication are early events in BE progression to EA, but reliable diagnostic assays to detect chromosomal markers in premalignant stages of EA arising from BE are lacking. Previously, we investigated chromosomal changes in an in vitro model of acid and bile exposure-induced Barrett’s epithelial carcinogenesis (BEC). In addition to detecting changes already known to occur in BE and EA, we also reported a novel recurring chromosomal translocation t(10:16) in the BE cells at an earlier time point before they undergo malignant transformation. In this study, we refine the chromosomal event with the help of fluorescence microscopy techniques as a three-way translocation between chromosomes 2, 10, and 16, t(2:10;16) (p22;q22;q22). We also designed an exclusive fluorescent in situ hybridization for esophageal adenocarcinoma (FISH-EA) assay that detects these chromosomal breakpoints and fusions. We validate the feasibility of the FISH-EA assay to objectively detect these chromosome events in primary tissues by confirming the presence of one of the fusions in paraffin-embedded formalin-fixed human EA tumors. Clinical validation in a larger cohort of BE progressors and non-progressors will confirm the specificity and sensitivity of the FISH-EA assay in identifying malignant potential in the early stages of EA.

Passage-Dependent Cancerous Transformation Of Human Mesenchymal Stem Cell Of Adipose Origin During In Vitro Culture

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4862-4862
Author(s):  
Jung Ah Kim ◽  
Qute Choi ◽  
Kyong Ok Im ◽  
Ji Seok Kwon ◽  
Si Nae Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Telomere Length ◽  
Conflicts Of Interest ◽  
Bac Clone ◽  
Interphase Cell ◽  
Cytogenetic Changes ◽  
Chromosomal Changes

Abstract Introduction In vitro culture of adult human mesenchymal stem cells (hMSCs) can induce cancerous transformation, depending on environmental factors. To evaluate the passage dependent chromosomal changes of hMSCs toward malignant transformation, we passaged adipose origin hMSC to 9th passage and analyzed cytogenetic change, molecular cytogenetic changes, and telomere length variations on every passage. Methods On each passage, in situ karyotyping was performed on 3 separate batches with subsequent Giemsa staining. Karyotyping was analyzed using Metafer system (MetaSystems, Altlussheim, Germany). For analysis of nonproliferating interphase cell, each chromosome were counted with centromere fluorescent in situ hybridization (FISH) using Same Day OligoFISH™(Cellay Inc., Cambridge, Massachusetts, USA). Telomere length was analyzed using FISH technique with a Cy3-lableled Telomere peptide nucleic acid (PNA) FISH kit (DakoCytomation Denmark A/S, Glostrup, Denmark). To confirm the chromosomal translocation appeared by in situ karyotyping, we made home-brew FISH probe with bacterial artificial chromosome(BAC) clone and quantitated the proportion of abnormal cells by interphase FISH. Results On 5th passage, translocation and polysomy of chromosome 7 and 9 appeared, and on 6th passage, additional translocation t(6; 10) appeared. ISCN Karyotypes of chromosomal changes from 5th passage to 7th passage were 48,XX,+7,t(7;22)(q11.22;q13.3),+9[4]/46,XX[21] → 47,XX,+7[2]/47,XX,t(6;10)(q21;q25.1),+7[2]/46,XX[13] → 48,XX,+7,t(7;22)(q11.22;q13.3),+9[6]/ 47,XX,+7[5]/46,XX[9]. Telomere length was decreased on late passages. Fusion signal of t(7;22) on passage 5(fig 1) and that of t(6;10) on passage 6(fig 2) were confirmed by BAC clone. Conclusions The behavior of late passage (from passage 5) follows a cytogenetic profile similar to that of transformed cancer cells. Cytogenetic abnormalities which were not observed in earlier passage, showed up and disappeared, but eventually persisted during passages. We suggest in vitro environment cause hMSCs to undergo cancer-like cytogenetic changes. It is of great importance to test safeguards for clinical applications of human stem cells manufactured in vitro. Disclosures: No relevant conflicts of interest to declare.

Early detection of mammary tumors in vivo using a highly specific tumor antibody.

2015 ◽  
Vol 33 (28_suppl) ◽  
pp. 14-14
Author(s):  
Didier Dréau ◽  
Laura Jeffords Moore ◽  
Lopamudra Das Roy ◽  
Shu-ta Wu ◽  
Rahul Puri ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Early Detection ◽  
Tumor Progression ◽  
T Antigen ◽  
Great Promise ◽  
Specificity And Sensitivity ◽  
Genetically Engineered Mice

14 Background: Earlier detection of abnormal cells using target-specific techniques holds great promise. One of those targets Mucin-1 (MUC1) is expressed as a hypo-glycosylated form (i.e., tMUC1) in 95% of breast tumors and plays a crucial role in cancer progression. We have developed Tab004, a monoclonal antibody highly specific to a protein sequence accessible in tMUC1. Here we present data assessing the specificity and sensitivity of Tab004 in vitro and in genetically engineered mice in vivo overtime. Methods: We have developed human MUC1 Tg mice that were bred to mice that carry the oncogene, polyoma middle T antigen driven by the MMTV promoter. These mice designated MMT develop mammary gland tumors spontaneously and expresses the human form of tMUC1. PyVMT and C57Bl/6 served as controls. Mice develop mammary gland hyperplasia between 8-10 weeks of age that progresses to ductal carcinoma in situ by 12-14 weeks and adenocarcinoma by 18-24 weeks. Approximately 40% of the mice develop metastasis to the lung and other organs. The tumor progression appropriately mimics the human disease. MMT mice (n = 20) were injected twice monthly retro-orbitally with 12.5ug (100uL) of Tab004-conjugated to indocyanine green (ICG) and imaged thereafter from 8 to 22 weeks of age. Fluorescence was assessed 4 and 24 hrs post injection using the IVIS system. At euthanasia, tissue was collected for further analyses. Further, human breast tumor and normal mammary epithelial tissues were evaluated by immunohistochemical staining. Results: Tab004 specifically recognizes tMUC1 and not normal MUC1. In MMT mice, ICG-conjugated Tab004 allowed early detection of tumors in vivo sparing recognition of normal mammary epithelia in the C57BL/6 mice or in the PyV MT tumors. Detection with ICG-conjugated Tab004 allowed monitoring of tumor progression overtime. Importantly, ICG-conjugated Tab004 permitted significantly earlier detection than physical examination. Conclusions: The data highlight the specificity and the sensitivity of Tab004 in detecting tMUC1 in vitro, in situ and in relevant murine models in vivo. Thus, Tab004 will have significant clinical relevance for development as a targeted imaging agent and in the future for targeted drug delivery.

PDL-1 and immunohistochemistry markers in esophageal adenocarcinoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e15567-e15567
Author(s):  
Dawn E. Jaroszewski ◽  
Joanne Xiu ◽  
Zoran Gatalica ◽  
Staci Beamer ◽  
Melissa Stanton ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Esophageal Adenocarcinoma ◽  
Neoadjuvant Treatment ◽  
Formalin Fixed Paraffin ◽  
Ffpe Tumor ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

e15567 Background: Esophageal adenocarcinoma (EAC) prognosis is poor and there is a need to identify patients that benefit most from neoadjuvant therapy. To examine the association of various biomarkers with clinical outcomes in neoadjuvant treatment of EAC, we retrospectively evaluated the biomarker expression (TS, ERCC1, TOPO1, PD-L1, PD-1) in patient matched formalin-fixed paraffin-embedded (FFPE) tumor samples. Methods: Immunohistochemistry of TS (TS106/4H4B1) , ERCC1 (Ab. 8F1), TOPO1 (1D6), PD-L1 (both 22c3 and SP142), PD-1 (NAT105), and chromogenic in-situ hybridization (CISH) of Her2 were performed on FFPE samples from 35 patients across 2 institutions at time of EAC diagnosis and after treatment when available. Retrospective clinical data and survival (5/2006-1/2016) was analyzed with a mean follow up of 110 months (range 22-306). Results: Overexpression (pre/post-treatment) of TS (60%/54%), ERCC1 (69%/16%), TOPO1 (74%/50%), PD-1 (54%/63%), PD-L1 (SP142) (2.9%/4%), PD-L1 (22c3) (0%/4%) and amplification of Her2 (18%/23%) were observed. Pretreatment observed PD-L1 levels were lower in our study (3%) when compared to other studies in EAC specimens (35%). Immunohistochemistry and changes observed after chemoradiation are reviewed in Table. No markers had significant correlation with prognosis however TS negative expression showed a non-significant (p=0.15) trend towards improved survival. Conclusions: Analyzing biomarkers in our neoadjuvant EAC cohort demonstrated a lower than expected PD-L1 positivity. In the largest cohort, to our knowledge, of patient matched FFPE tumor samples, we did not observe a statistically significant association between TS, ERCC1, TOPO1, PD-L1, or PD-1 with improved clinical outcomes. [Table: see text]

Detection of chromosomal changes in chronic lymphocytic leukemia using classical cytogenetic methods and FISH: application of rich mitogen mixtures for lymphocyte cultures

2016 ◽  
Vol 64 (4) ◽  
pp. 894-898 ◽  
Author(s):  
Dorota Koczkodaj ◽  
Sylwia Popek ◽  
Szymon Zmorzyński ◽  
Ewa Wąsik-Szczepanek ◽  
Agata A Filip
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mitotic Index ◽  
Cell Cultures ◽  
Lymphocytic Leukemia ◽  
Pokeweed Mitogen ◽  
Structural Aberrations ◽  
Chromosomal Changes

One of the research methods of prognostic value in chronic lymphocytic leukemia (CLL) is cytogenetic analysis. This method requires the presence of appropriate B-cell mitogens in cultures in order to obtain a high mitotic index. The aim of our research was to determine the most effective methods of in vitro B-cell stimulation to maximize the number of metaphases from peripheral blood cells of patients with CLL for classical cytogenetic examination, and then to correlate the results with those obtained using fluorescence in situ hybridization (FISH). The study group involved 50 consecutive patients with CLL. Cell cultures were maintained with the basic composition of culture medium and addition of respective stimulators. We used the following stimulators: Pokeweed Mitogen (PWM), 12-O-tetradecanoylphorbol 13-acetate (TPA), ionophore, lipopolysaccharide (LPS), and CpG-oligonucleotide DSP30. We received the highest mitotic index when using the mixture of PWM+TPA+I+DSP30. With classical cytogenetic tests using banding techniques, numerical and structural aberrations of chromosomes were detected in 46 patients, and no change was found in only four patients. Test results clearly confirmed the legitimacy of using cell cultures enriched with the mixture of cell stimulators and combining classical cytogenetic techniques with the FISH technique in later patient diagnosing.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

CONSERVATION OF RARE SPECIES PLANTS AND LICHENS IN SITU WITH PLANNED ANTHROPOGENIC ACTIVITIES: BASIC APPROACHES AND IMPLEMENTATION PRINCIPLES

2018 ◽  
Vol 12 (7-8) ◽  
pp. 38-45
Author(s):  
A. N. EFREMOV ◽  
N. V. PLIKINA ◽  
T. ABELI
Keyword(s):  
Rare Species ◽  
Technology Implementation ◽  
Technology Development ◽  
Anthropogenic Activities ◽  
Natural Habitat ◽  
Local Population ◽  
Main Stage ◽  
Social Risks

Rare species are most vulnerable to man-made impacts, due to their biological characteristics or natural resource management. As a rule, the economic impact is associated with the destruction and damage of individual organisms, the destruction or alienation of habitats. Unfortunately, the conservation of habitat integrity is an important protection strategy, which is not always achievable in the implementation of industrial and infrastructural projects. The aim of the publication is to summarize the experience in the field of protection of rare species in the natural habitat (in situ), to evaluate and analyze the possibility of using existing methods in design and survey activities. In this regard, the main methodological approaches to the protection of rare species in the natural habitat (in situ) during the proposed economic activity were reflected. The algorithm suggested by the authors for implementing the in situ project should include a preparatory stage (initial data collection, preliminary risk assessments, technology development, obtaining permitting documentation), the main stage, the content of which is determined by the selected technology and a long monitoring stage, which makes it possible to assess the effectiveness of the taken measures. Among the main risks of in situ technology implementation, the following can be noted: the limited resources of the population that do not allow for the implementation of the procedure without prior reproduction of individuals in situ (in vitro); limited knowledge of the biology of the species; the possibility of invasion; the possibility of crossing for closely related species that сo-exist in the same habitat; social risks and consequences, target species or population may be important for the local population; financial risks during the recovery of the population. The available experience makes it possible to consider the approach to the conservation of rare species in situ as the best available technology that contributes to reducing negative environmental risks.

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