Fluorescence in Situ Hybridization (FISH)

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Neuropathology for the Neuroradiologist: Fluorescence in Situ Hybridization

Neuropathology for the Neuroradiologist: Fluorescence in Situ Hybridization

T he field of molecular cytogenetics has opened an exciting avenue of discovery by using chromosome analysis to identify clinically useful diagnostic, prognostic, and predictive biomarkers. Neuropathologists have exploited this informa- tion with a number of molecular techniques, one of which is known as fluorescence in situ hybridization (FISH) to subtype phenotypically similar neoplasms and to unlock the genetic forces that drive tumor development. Appreciation of this tool can enhance the interactions of the neuroradiologist with the neuropathologist and deepen understanding of commonly occurring tumors beyond standard histologic recognition. The purpose of this report is to review the significance of FISH in the context of diagnostic tumor pathology.

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Multicolor banding remains an important adjunct to array CGH and conventional karyotyping

Multicolor banding remains an important adjunct to array CGH and conventional karyotyping

Multicolor banding (MCB) is a highly reliable and repro- ducible fluorescence in situ hybridization (FISH) tech- nique that can be used for the detection of balanced and unbalanced chromosome rearrangements and to help characterize complex chromosome rearrangements and marker chromosomes [1,2] that cannot be characterized by array comparative genomic hybridization (CGH) or by G-banded chromosome analysis and standard FISH techniques alone. Images are processed using specialised software (MetaSystems - Isis) which converts the fluor- escence profiles along the length of the chromosome to

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Molecular cytogenetic identification of three rust-resistant wheat-Thinopyrum ponticum partial amphiploids

Molecular cytogenetic identification of three rust-resistant wheat-Thinopyrum ponticum partial amphiploids

Background: Thinopyrum ponticum (2n = 10× = 70, J S J S J S J S JJJJJJ) is an important wild perennial Triticeae species that has a unique gene pool with many desirable traits for common wheat. The partial amphiploids derived from wheat-Th. ponticum set up a bridge for transferring valuable genes from Th. ponticum into common wheat. Results: In this study, genomic in situ hybridization (GISH), multicolor GISH (mcGISH) and fluorescence in situ hybridization (FISH) were used to analyze the genomic constitution of SN0389, SN0398 and SN0406, three octoploid accessions with good resistance to rust. The results demonstrated that the three octoploids possessed 42 wheat chromosomes, while SN0389 contained 12 Th. ponticum chromosomes and SN0398 and SN0406 contained 14 Th. ponticum chromosomes. The genomic constitution of SN0389 was 42 W + 12J S , and for SN0398 and SN0406 it was 42 W + 12J S + 2 J. Chromosomal variation was found in chromosomes 1A, 3A, 6A, 2B, 5B, 6B, 7B, 1D and 5D of SN0389, SN0398 and SN0406 based on the FISH and McGISH pattern. A resistance evaluation showed that SN0389, SN0398 and SN0406 possessed good resistance to stripe and leaf rust at the seedling stage and adult-plant stage.

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Applications of Fluorescence in Situ Hybridization (FISH) for Detecting Genetic Changes in Hematological Malignancies

Applications of Fluorescence in Situ Hybridization (FISH) for Detecting Genetic Changes in Hematological Malignancies

with additional changes), +21(25%), del(17)(12.5%), and monosomy 9(12.5%) by interphase FISH. We suggest that all these aberrations, occurring in 75% of AML cases, 12.5% MDS and 12.5% of ALL with secondary changes, should be denoted major route abnormalities. Among the approximately 1600 CML cases with standard Ph and secondary changes published to date [33], +8, +Ph, i(17q) and +19 have been described in 35, 31, 21, and 14% of the cases, respectively. The incidence of trisomy 8 in 140 Chinese patients with CLL were found in only two patients (1.4%) [34]. Our study demonstrates that trisomy 8 was not found in CLL, and its role in prognosis of CLL remains unknown. Using conventional cytogenetic methods in which 20 to 30 metaphase cells are typically examined, it is possible to find 2 or more metaphases with trisomy 8 in many of these patients. According to the ISCN definition, 6 these specimens would have an abnormal clone. In our study, +8, − 9 and +21 are clonal by ISCN criteria and may not reflect either artifact or the potential emergence of an abnormal clone. All other additional chromosomal abnormalities occur in less than 10% of the CML cases, the most frequent being –Y, +21, +17, –7, and –17 [35]. Anastasi et al. [36] demonstrated that of three patients with MDS associated with trisomy 8. Structural rearrange- ments involving duplication of 21q, dup(21q) have also been described [27-29]. Combining these myeloid disorder groups and our findings, the most common additional chromosomal changes are +8, +Ph, i(17q), del(17), +19, –Y, +21, +17, –9 and –7. These abnormalities were proposed to follow the major route of clonal evolution, whereas other changes evolving more rarely were suggested to follow the minor route [37]. In view of our present knowledge, it may be reasonable to expand the major evolutionary route to include all these aberrations, using 5% as a reasonable cut-off value. Our study shows that FISH analysis using directly labeled DNA probes specific for chromosome 8 is an excellent way to detect trisomy 8 cells in hematologic malig- nancies. iFISH could also be used to monitor the effects of treatment, in patients found to have trisomy 8 at the time of diagnosis, or to detect early relapse. We believe this study predicts that such analyses could be extended to other chromosomal trisomies and monosomies; although the latter will require even stricter attention to normal value studies.

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Case Report Richter transformation with c- MYC overexpression: report of three cases

Case Report Richter transformation with c- MYC overexpression: report of three cases

Abstract: Pathogenesis of Richter transformation (RT) or Richter syndrome (RS) of chronic lymphocytic leukemia (CLL) is still largely unknown. Increasing evidences show that c-MYC may play a role in the development of RS. Here we report three cases of RS with overexpression of c-MYC. The first case was a 78-year-old male who initially pre- sented with CLL and then developed diffuse lymphadenopathy and ascites shortly after. Ascites cytology showed a population of large lymphoid cells positive for MYC (8q24) rearrangement by fluorescence in situ hybridization (FISH) and overexpression of c-MYC by immunohistochemistry (IHC). The second case was a 66-year-old male presented with rapidly enlarging lymph nodes and pleural effusion after a long history of CLL. Biopsy showed large B-cells posi- tive for c-MYC overexpression and high Ki-67 proliferation index (80-90%). The third case was a 62-year-old female with CLL who presented for lobectomy for lung adenocarcinoma. Interestingly, along with the carcinoma, large B-cell lymphoma was incidentally found which had the same immunophenotype as the CLL. FISH analysis revealed gain of c-MYC at 8q and IHC showed increased c-MYC expression. This study supports that c-MYC plays a critical role in RS.

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Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures

Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures

TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontubercu- lous mycobacteria (NTM) in acid-fast bacillus-positive (AFB ⴙ ) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB ⴙ cultures for microscopic examination. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB ⴙ cultures from Denmark and 42 AFB ⴙ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies. The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of Mycobacterium fortuitum, which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC (n ⴝ 46) or NTM (n ⴝ 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%.

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Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH)

Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH)

Fluorescence in  situ hybridization (FISH) has been widely used as a cultivation-independent tool for direct detection, identification and quantification of microor- ganisms [16]. The cultivation-independent characteristic of this technique is also particularly important for micro- organisms such as obligate biotrophs, as it allows for the direct study of plant pathogens in their natural environ- ment [17, 18]. Although updated techniques, applications and protocol improvements are now available for FISH, the technique is based around four core steps: (1) speci- men fixation and immobilization; (2) permeabilization to increase accessibility of an organism specific-nucleic acid probe to the target; (3) hybridization of the probe; (4) washing to remove unbound probe; and (4) documenta- tion by microscopy or flow cytometry [16, 19, 20]. Typi- cal oligonucleotide probes used for FISH range between 15 and 30 base pairs in length and are labeled with one or more fluorescent dyes [20]. Most FISH applications target ribosomal RNA (rRNA), as these molecules are highly abundant and stable within cells, and possess both variable and highly conserved sequence domains [19]. Single copy genes can also be detected using FISH when coupled with signal amplification techniques such as catalyzed reporter deposition—CARD-FISH [21]. Even though FISH assays using oligonucleotide probes tar- geting rRNA were first introduced almost 30  years ago [22], only few studies have applied this technique for the visualization of oomycete plant pathogens such as Phy- tophthora agathidicida and P. cinnamomi [23–25]. Non- specific fluorescent staining techniques have been used to visualize infection structures and plant cellular growth and response to the grape downy mildew pathogen Plas- mopara viticola [26–28], and to visualize the in planta development of pathogens such as Peronospora sparsa, Pe. tabacina, Pseudoperonospora cubensis, and Ps.

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16p13.3 duplication associated with non-syndromic pierre robin sequence with incomplete penetrance

16p13.3 duplication associated with non-syndromic pierre robin sequence with incomplete penetrance

To investigate whether the duplicated 14q11.2 and pter-16p13.13 regions played a role in PRS development, all cases with comparable chromosome 14q and 16p du- plication documented in the sSMC database [20] and PubMed were reviewed. A total of ten cases with sSMC at the breakpoint of 14q11.2 were identified in the data- base [20] (Table 1). In these ten sSMC cases, cytogeneti- cally, three of them had ring-shaped sSMCs (cases 4, 7, and 8), three of them had minute-shaped sSMCs (cases 3, 9, and 10), two of them were dicentric (cases 1 and 2), and the remaining two were inverted duplicated sSMCs (cases 5 and 6). Mosaicism was presented in three cases (cases 2, 3, and 8). The identification and characterization methods of sSMCs included FISH, array CGH, and multi- plex ligation-dependent probe amplification (MLPA). Six cases were characterized by FISH methods including cen-FISH, multiplex fluorescence in situ hybridization (M-FISH), and various FISH probes (cases 1, 2, 3, 5, 8, and 9).

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Acute EBV infection masquerading as "In-situ Follicular Lymphoma": a pitfall in the differential diagnosis of this entity

Acute EBV infection masquerading as "In-situ Follicular Lymphoma": a pitfall in the differential diagnosis of this entity

We present the case of a 30 year-old man who was referred for evaluation of diffuse lymphadenopathy. Six weeks prior, he noticed darkening of his urine associated with pale stools, nausea and an eventual 30 lb weight loss within a month. The initial laboratory findings showed elevation of the liver enzymes. A CT scan showed mesenteric and periaortic lymphadenopathy with the largest lymph node measuring 2.8 cm. Other laboratory results were otherwise unremarkable (including a normal LDH) with the exception of positive serum antibodies against Epstein-Barr virus (EBV) associated antigens (IgM+ and IgG+). An excisional biopsy of 4 of the small neck lymph nodes showed a normal architecture with prominent follicles and an intact capsule. But, by immunohistochemistry two of the follicles showed aberrant coexpression of BCL-2, in addition to CD10 and BCL-6. In-situ hybridization for early Epstein-Barr virus mRNA (EBER) and immunohistochemistry for latent membrane protein-1 (LMP-1) stained both scattered positive cells, as well as BCL-2 positive B-cells. Although an original diagnosis of in-situ follicular lymphoma was favored at an outside facility, additional interphase fluorescence in situ hybridization (FISH) studies for t(14;18);(IGH-BCL2) rearrangement (performed on the BCL-2 + follicles microdissected from the tissue block; Abott probe dual colour fusion) and molecular studies (IGH gene rearrangement by PCR, also performed on the microdissected follicles) were negative. Serologic studies (positive EBV antibodies) and immunostains in conjunction with the molecular studies confirmed the reactive nature of the changes. Our case also shows direct immunopathogenic evidence of BCL-2 expression among the EBV-infected cells, which has to our knowledge not been previously documented in vivo. A diagnosis of EBV infection should, therefore, be considered when confronted with BCL-2 expression in germinal centers, particularly in younger individuals, as the diagnosis of FLIS may lead to extensive and invasive haematologic work-ups.

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Familiar unbalanced complex rearrangements involving 13 p-arm: description of two cases

Familiar unbalanced complex rearrangements involving 13 p-arm: description of two cases

We had the opportunity to study two non-traditional rearrangements that gave rise to partial trisomies with- out an apparent phenotypic effect. The first case showed a translocation involving chromosomes 4qter and 13p and the second involving chromosomes 7qter and 13p. In both cases, conventional cytogenetics analysis showed normal chromosomes, the fluorescence in situ hybridization (FISH) study with specific subtelomeric probes evidenced a third signal on chromosome 13p and finally genomic study by array comparative genomic hybridization (CGH) defined the triple dose segment.

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Molecular cytogenetic characterization of undifferentiated embryonal sarcoma of the liver: a case report and literature review

Molecular cytogenetic characterization of undifferentiated embryonal sarcoma of the liver: a case report and literature review

detected in the MALAT1 gene, i.e., the MALAT1 gene was rearranged at t(11;19) [9]. Point mutations of the TP53 gene were also detected in three UESL cases [10,11]. To our knowledge, we are the first to use high-resolution array-CGH analysis (aCGH) to detect more subtle seg- mental genomic imbalances, which were followed by fluorescence in situ hybridization (FISH) to confirm some of the selected anomalies identified by aCGH. In addition, given that a hemizygous deletion of the TP53 gene was identified in our case by aCGH and FISH, subsequent DNA sequencing was performed to identify additional gene mutations in the rest of the TP53 allele in this case.

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Analphoid supernumerary marker chromosome characterized by aCGH and FISH as inv dup(3)(q25.33qter) de novo in a child with dysmorphic features and streaky pigmentation: case report

Analphoid supernumerary marker chromosome characterized by aCGH and FISH as inv dup(3)(q25.33qter) de novo in a child with dysmorphic features and streaky pigmentation: case report

organizer region (NOR) staining negative. Fluorescence in situ hybridization studies using Pan-centromeric alpha- satellite FISH probe (Cambio) showed no hybridization onto the marker chromosome (Fig. 1b), confirming it to be analphoid. Immuno-FISH studies for centromere spe- cific proteins could not be undertaken to confirm the for- mation of a neocentromere. However, the fact that the marker was confirmed as analphoid by FISH and was also found to be highly stable in skin fibroblast culture, strongly suggest of a possible neocentromere formation. Peripheral blood chromosomes of the parents were nor- mal by G-banding and by all chromosome specific sub- telomeric FISH analysis. Array-CGH studies on cultured skin fibroblast cells of the patient showed amplification of oligo-probes from probe FLJ14153 at 3q25.33 (160.03 Mb) to 3qter (199.29 Mb) (Fig. 2) confirming the origin and breakpoint of the marker chromosome. The observa- tion was further validated by FISH studies using chromo- some 3 specific subtelomere FISH probe (Vysis), which revealed the marker to be inversion-duplication 3q25.33- qter (Fig. 1c).

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Determination of Telomere Length by the Quantitative Fluorescence in Situ Hybridization (Q FISH) Method

Determination of Telomere Length by the Quantitative Fluorescence in Situ Hybridization (Q FISH) Method

Telomeres are nucleoprotein complexes located at the ends of eukaryotic chromosomes and are shorten with aging or various causes. Shortened telomere plays an important role for chromo- somal instability in carcinogenesis, or a number of diseases in relation to aging. FISH using pep- tide-nucleic acid (PNA) probe is suitable for analyzing telomeres, because telomere is a part of DNA molecule and localized in chromosomal end in loop shape structure (T-loop). We introduce our telomere analysis for tissue sections and cultured cells in this paper. On the tissue sections, we analyze telomere length with telomere intensity to centromere intensity ratio (TCR), centromere as an inner control. In the cultured cells, we analyze telomere lengths on each chromosome with karyotyping. Those Q-FISH methods by PNA probe are an accurate and reliable tool for research on telomere lengths in various conditions in biology.

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Evaluation of the accuracy of FISH plus conventional fetal karyotyping in a University Hospital in China

Evaluation of the accuracy of FISH plus conventional fetal karyotyping in a University Hospital in China

The conventional cytogenetic banding of metaphase chromosomes in cultured amniotic fluid amniocytes is the golden standard for the detection of numerical and structural chromosomal aberrations (Lo YM. 2012; Jobanputra et al., 2002; kale et al., 2014). In addition, rapid aneuploidy detection (RAD) methods such as fluorescence in situ hybridization (FISH), which enumerate chromosomes 13, 18, 21, X, and Y of interphase chromosomes from uncultured amniotic fluid amniocytes usually completes within 48h, has been regarded as an adjunct method of karyotyping and it is now wide spread used (Ho et al., 2012; Jia et al., 2011; Elsayed et al., 2013).

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Array-based comparative genomic hybridization is more informative than conventional karyotyping and fluorescence in situ hybridization in the analysis of first-trimester spontaneous abortion

Array-based comparative genomic hybridization is more informative than conventional karyotyping and fluorescence in situ hybridization in the analysis of first-trimester spontaneous abortion

Based on knowledge about the frequencies of specific trisomies in spontaneous abortions and the availability of commercial multiplex probe sets, FISH was per- formed using three multicolor probe mixtures (GP Med- ical Technologies Inc., Beijing, China). Probe mix 1 and mix 2 contain locus-specific probes that identify chro- mosomes 13, 21 and 16, 22 respectively. Probe mix 3 contains centromere site probes that identify chromo- somes 18, X and Y. Each probe set was applied to one of the two slides from each case. The probes were direct- labeled with different fluorophores, which could be visualized with appropriate filter combinations. Hybridization and post-washing conditions were per- formed according to the manufacturer’s instructions. Slides were counterstained with 15 ml of a very dilute 4,6-diamidino-2-phenylindole antifade solution for 10 minutes and observed under a fluorescent microscope (BX51, Olympus, Tokyo, Japan) equipped with an appro- priate filter.

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Assessment of ERBB2 and EGFR gene amplification and protein expression in gastric carcinoma by immunohistochemistry and fluorescence in situ hybridization

Assessment of ERBB2 and EGFR gene amplification and protein expression in gastric carcinoma by immunohistochemistry and fluorescence in situ hybridization

transmembrane segment (aa 633-654), and a cytoplas- mic domain that exhibits tyrosine kinase activity (aa 655-1234) [2,3], ERBB2 is frequently unregulated in human cancers such as breast cancer [4], ovarian cancer [5], and so on. High expression level of ERBB2 has been significantly correlated with increased tumor invasion, metastasis, resistance to chemotherapy, and poor prog- nosis of patients [6]. However, a lack of reliable and accurate methods to assess the relationship between EGFR and ERBB2 gene status and protein expression in gastric cancer has limited correlative assessments with clinical parameters, including survival and sensitivity to targeted agents. Therefore, in the present study, we uti- lized fluorescence in situ hybridization (FISH) to assess amplification of the ERBB2 and EGFR genes in gastric cancer patient samples. We combined this data with results from histopathological and immunohistochemical analyses to determine the relationship between ERBB2

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Target fluorescence in-situ hybridization (Target FISH) for plasma cell enrichment in myeloma

Target fluorescence in-situ hybridization (Target FISH) for plasma cell enrichment in myeloma

It is not recommended to perform interphase FISH directly on the PCM bone marrow due to often low plasma cell percentage and admixture by other hemopoietic cells. The plasma cells should be selected either by immuno- magnetic beads or flow cytometry based plasma cell sorting or concurrent labeling of the cytoplasmic immunoglobulin (cIg) light chain to allow unambiguous detection of cyto- genetic abnormalities in the neoplastic plasma cell popula- tion[3, 4]. The enrichment techniques of cell sorting [5] or cIg-FISH [6] however are labor, time and cost intensive. Hence the incorporation of these methods into the routine workflow of a diagnostic cytogenetics laboratory may be challenging. An alternative method of plasma cell enrich- ment termed Target FISH based on sequential MGG stain to identify plasma cell populations to be followed by FISH analysis was previously reported [7]. Herein, we present an automated Target FISH system for use in routine molecular diagnostics.

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Case Report Primitive myxoid mesenchymal tumor of the infancy in abdominal cavity: report of a rare case

Case Report Primitive myxoid mesenchymal tumor of the infancy in abdominal cavity: report of a rare case

Abstract: Primitive myxoid mesenchymal tumor of the infancy (PMMTI) is an extremely rare soft tissue tumor. We report a case of 2-year-old girl presenting with omentum and intestinal wall involvement in abdominal cavity with prominent refractory ascites. Histologically, the tumor consisted of primitive spindle or polygonal cells dispersing in a myxoid background with delicate blood vessels. Immunohistochemically, tumor cells expressed vimentin and NSE but were negative for AE1/AE3, CK5/6, TTF1, desmin, SMA, MyoD1, myogenin, ALK, CD34, calretinin, WT1, HBME- 1, D2-40, S-100 protein, CD56, synaptophysin, CD99, CD20, CD3, CD30. Fluorescence in situ hybridization (FISH) showed absences of chromosome translocation involving DDIt3, EWSR1 and EtV6. The patients undergone a pal- liative surgery and succumbed after 2 months. To our knowledge, this is the first case of PMMTI affecting abdominal cavity and demonstrating a more unfavorable prognosis.

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Genetic analysis of products of conception using a HLPA/SNP-array strategy

Genetic analysis of products of conception using a HLPA/SNP-array strategy

G-banded karyotyping is the conventional cytogenetic technique used in analysis of products of conception (POC), which could detect chromosomal aneuploidies, structural abnormalities, duplications or deletions (>5- 10Mb), polyploidies and mosaicism. However, it has several limitations such as low resolution, high rates of culture fail- ure and long reporting time [4]. Then some rapid tech- niques for genetic testing of chromosomal aneuploidies have emerged including quantitative fluorescent PCR (QF-PCR), fluorescence in situ hybridization (FISH),

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Case Report Diffuse large B-cell lymphoma of the uterus suspected of having transformed from a marginal zone B-cell lymphoma harboring trisomy 18: a case report and review of the literature

Case Report Diffuse large B-cell lymphoma of the uterus suspected of having transformed from a marginal zone B-cell lymphoma harboring trisomy 18: a case report and review of the literature

Abstract: The patient was a 72-year-old female with the chief complaint of abdominal fullness. A giant primary myoma of the uterine cervix was suspected, and total hysterectomy was performed. Based on a postoperative histo- pathological examination of the tumor a diagnosis of diffuse large B-cell lymphoma (DLBCL) was made in the uterus and a mass in the greater omentum was diagnosed as a marginal zone B-cell lymphoma (MZBCL). No flow-cytometry studies or chromosome or gene examinations were performed on a fresh specimen. The results of an examination of a paraffin block histopathology specimen by fluorescence in-situ hybridization (FISH) showed no mucosa associ- ated lymphoid tissue lymphoma translocation gene 1 (MALT1) (18q21.1), B-cell lymphoma 2 (BCL2) (18q21.3), or BCL6 (3q27) split signals in either the uterus or the greater omentum, however, trisomy 18 was detected in approxi- mately 50%-70% of the tumor cells in both the uterus and the greater omentum. Trisomy 18 was present in around 15-33% of the DLBCL cells and MZBCL cells. These findings suggested a strong possibility that the tumor cells in the uterus and greater omentum were the same clone and that transformation from MZBCL to DLBCL had occurred. Since DLBCLs that result from a transformation usually have a worse outcome than de novo DLBCLs, even when a DLBCL seems to have originated in the uterus the surrounding tissue should always be examined, and caution should be exercised in regard to transformation from a low-grade B-cell lymphoma to a DLBCL.

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