scholarly journals Establishment of multiplex RT-PCR to detect fusion genes for the diagnosis of Ewing sarcoma

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Hitomi Ueno-Yokohata ◽  
Hajime Okita ◽  
Keiko Nakasato ◽  
Chikako Kiyotani ◽  
Motohiro Kato ◽  
...  
Keyword(s):  
Ewing Sarcoma ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Terminal Region ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Generation Sequencing

Abstract Background Detection of the tumor-specific EWSR1/FUS-ETS fusion gene is essential to diagnose Ewing sarcoma. Reverse transcription–polymerase chain reaction (RT–PCR) and fluorescence in situ hybridization are commonly used to detect the fusion gene, and assays using next-generation sequencing have recently been reported. However, at least 28 fusion transcript variants have been reported, making rapid and accurate detection difficult. Methods We constructed two sets of multiplex PCR assays and evaluated their utility using cell lines and clinical samples. Results EWSR1/FUS-ETS was detected in five of six tumors by the first set, and in all six tumors by the second set. The fusion gene detected only by the latter was EWSR1-ERG, which completely lacked exon 7 of EWSR1. The fusion had a short N-terminal region of EWSR1 and showed pathologically atypical features. Conclusions We developed multiplex RT–PCR assays to detect EWSR1-ETS and FUS-ETS simultaneously. These assays will aid the rapid and accurate diagnosis of Ewing sarcoma. In addition, variants of EWSR1/FUS-ETS with a short N-terminal region that may have been previously missed can be easily detected.

scholarly journals Nanostring-based screening for tyrosine kinase fusions in inflammatory myofibroblastic tumors

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Taisei Kurihara ◽  
Yoshiyuki Suehara ◽  
Keisuke Akaike ◽  
Takuo Hayashi ◽  
Shinji Kohsaka ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Rna Sequencing ◽  
Fusion Gene ◽  
Rt Pcr ◽  
Anaplastic Lymphoma ◽  
Inflammatory Myofibroblastic Tumors ◽  
Polymerase Chain ◽  
Lymphoplasmacytic Infiltration ◽  
Tyrosine Receptor Kinase

Abstract Gene expression imbalances were measured for tyrosine kinase (TK) genes using Nanostring in 19 samples of inflammatory myofibroblastic tumor (IMT). All cases were immunohistochemically stained with anaplastic lymphoma kinase (ALK) and pan-tropomyosin-related-kinase (pan-Trk) antibodies. Five cases with imbalanced ALK expression, reported with Nanostring, were tested using fluorescence in situ hybridization (FISH); two cases with imbalanced neurotrophic tyrosine receptor kinase 3 (NTRK3) expression were tested using reverse transcription-polymerase chain reaction (RT-PCR). One case with imbalanced expression for ROS proto-oncogene 1 (ROS1) was tested using RNA sequencing and RT-PCR. TK fusions were detected in all cases with imbalanced TK expression. RNA sequencing detected a FN1–ROS1 fusion gene in an adult IMT case. IMT with ALK rearrangement showed myofibroblast-dominant features. IMT with ETV6–NTRK3 fusion showed prominent lymphoplasmacytic infiltration with scattered myofibroblasts. Pan-Trk IHC revealed only scattered positively stained cells in IMT with ETV6–NTRK3 fusion gene. ROS1-positive IMT showed myofibroblast-dominant features.

Sensitive Detection of FIP1L1-PDGFRA Fusion Transcripts by Real-Time Quantitative RT-PCR (RQ-PCR) Reveals Achievement of Molecular Remission in Chronic Eosinophilic Leukemia Treated with Low-Dose Imatinib Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 483-483
Author(s):  
J. Jovanovic ◽  
A. Reiter ◽  
D. Cilloni ◽  
E. Gottardi ◽  
J. Score ◽  
...  
Keyword(s):  
Real Time ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Imatinib Therapy ◽  
Stem Cell Population ◽  
Rt Pcr ◽  
Molecular Remission ◽  
Assay Sensitivity ◽  
Chronic Eosinophilic Leukemia ◽  
Pcr Assays

Abstract The FIP1L1-PDGFRA fusion gene generated by a cryptic interstitial deletion at 4q12 is a recurrent molecular lesion in idiopathic hypereosinophilic syndrome (HES), that forms a basis for diagnosis of chronic eosinophilic leukemia (CEL). This disease entity is particularly important to recognize, since presence of the FIP1L1-PDGFRA fusion predicts a favorable response to molecularly targeted therapy in the form of imatinib, with clinical remissions being achieved with lower doses than are required in BCR-ABL+ chronic myeloid leukemia (CML). In order to improve our understanding of the biology of CEL and to provide a tool to improve the management of patients with this disorder we have developed real-time quantitative reverse transcriptase PCR (RQ-PCR) assays for the FIP1L1-PDGFRA fusion. Taking into account the marked heterogeneity in upstream breakpoints within the FIP1L1 gene, RQ-PCR assays were designed for cases with FIP1L1 breakpoints leading to fusion of exon 9, 10, 11, 12 or 13 to PDGFRA exon 12. FIP1L1-PDGFRA expression was compared to that of validated Europe Against Cancer endogenous control genes - β2microglobulin (B2M) and ABL. Serial dilution of the FIP1L1-PDGFRA+ EOL1 cell-line in fusion gene negative filler cells (HL60) revealed an assay sensitivity of 1 in 105. While identification of the FIP1L1-PDGFRA fusion using conventional RT-PCR approaches can be problematic, RQ-PCR analysis undertaken in diagnostic samples from 31 patients with FIP1L1-PDGFRA+ CEL (median age 53, 31–64 years) revealed that, in the majority, the fusion transcript was expressed at relatively high level (median deltaCt FIP1L1-PDGFRA - B2M, 12.2; median deltaCt FIP1L1-PDGFRA - ABL, 2.3). The FIP1L1-PDGFRA fusion was expressed at comparable level in blood and marrow at diagnosis of CEL. Serial monitoring was undertaken in 17 patients following initiation of imatinib 100mg/d. Overall, 8/8 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcript level within the first year of therapy. In follow-up samples affording a sensitivity of at least 1 in 1000, PCR negativity by quantitative and conventional nested RT-PCR was documented in 8/17 patients following a median of 21 weeks of imatinib (4–64 weeks); in two cases profound PCR negativity (i.e. at a sensitivity level of at least 1 in 105) was documented following 13 weeks and 2 years of imatinib, respectively. Overall, these data demonstrate that CEL with the FIP1L1-PDGFRA fusion is uniquely sensitive to imatinib therapy. This contrasts with BCR-ABL+ CML, in which molecular remission is generally not achieved - a phenomenon that has been postulated to reflect the persistence of a primitive quiescent stem cell population that is resistant to this agent. Understanding the biological basis for the differences in molecular response to imatinib in CML and CEL, may yield further improvements in molecularly-targeted therapeutic approaches.

scholarly journals A novel BCR-ABL fusion gene (e6a2) in a patient with Philadelphia chromosome-negative chronic myelogenous leukemia

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 2236-2240 ◽  
Author(s):  
A Hochhaus ◽  
A Reiter ◽  
H Skladny ◽  
JV Melo ◽  
C Sick ◽  
...  
Keyword(s):  
Southern Blot ◽  
Chronic Myelogenous Leukemia ◽  
Rare Variants ◽  
Fusion Gene ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Rt Pcr ◽  
Novel Variant ◽  
Polymerase Chain

A novel variant of the chimeric BCR-ABL mRNA transcript was detected in a patient with Philadelphia chromosome-negative (Ph-) chronic myelogenous leukemia (CML) by multiplex reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis of the fusion region of the amplified cDNA fragment showed an in-frame joining of exon e6 of the BCR gene and exon a2 of the ABL gene, giving rise to an e6a2 BCR-ABL transcript. This finding was confirmed by Southern blot analysis using a specific probe corresponding to intron 6 of the BCR gene, whereas conventional Southern blot for rearrangement of the major breakpoint cluster region (M-bcr) was negative. Western blot studies detected a BCR-ABL protein slightly larger than p185 BCR-ABL. Metaphase fluorescence in situ hybridization showed an insertion of ABL material into the BCR region without reciprocal BCR translocation. The findings in this case show that atypical BCR-ABL transcripts are detectable even in Ph- CML patients without M-bcr-rearrangements. Multiplex PCR using primers that allow for amplification of all known BCR-ABL transcripts is an appropriate method to exclude these rare variants.

A subset of multiple myeloma harboring the t(4;14)(p16;q32) translocation lacks FGFR3 expression but maintains anIGH/MMSET fusion transcript

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2374-2376 ◽  
Author(s):  
Madhumita Santra ◽  
Fenghuang Zhan ◽  
Erming Tian ◽  
Bart Barlogie ◽  
John Shaughnessy
Keyword(s):  
Plasma Cells ◽  
Early Stage ◽  
Fusion Transcript ◽  
Current Data ◽  
Rt Pcr ◽  
Microarray Profiling ◽  
Recurrent Translocation ◽  
Polymerase Chain ◽  
Multiple Myelomas

Previous studies have revealed that that approximately 10% to 15% of multiple myelomas (MMs) are characterized by a reciprocal t(4;14)(p16;q32) translocation that activates expression ofFGFR3 and creates an IGH/MMSET fusion transcript. Current data suggest that activation of FGFR3is the oncogenic consequence of this rearrangement. Using a combination of microarray profiling, reverse transcriptase–polymerase chain reaction (RT-PCR), and interphase fluorescence in situ hybridization (FISH), we show that 32 (18%) of 178 newly diagnosed cases of MM harbor the t(4;14)(p16;q32). Importantly, 32% of these cases lack expression of FGFR3, yet express MMSET and have an IGH/MMSET fusion transcript. Interphase FISH showed that whereas the IGH/MMSET fusion was present in more than 80% of the clonotypic plasma cells in these novel cases, there was typically a complete loss of one copy of FGFR3. These data indicate that the t(4;14)(p16;q32) and loss of FGFR3occurred at a very early stage and suggest that activation ofMMSET, not FGFR3, may be the critical transforming event of this recurrent translocation.

scholarly journals Detection of the chromosome 16 CBF beta-MYH11 fusion transcript in myelomonocytic leukemias

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1313-1322 ◽  
Author(s):  
H Poirel ◽  
I Radford-Weiss ◽  
K Rack ◽  
X Troussard ◽  
A Veil ◽  
...  
Keyword(s):  
Single Case ◽  
Fusion Transcript ◽  
Chromosome 16 ◽  
Rt Pcr ◽  
Fusion Transcripts ◽  
Prognostic Importance ◽  
Polymerase Chain

Karyotypic detection of chromosomal 16 abnormalities classically associated with AML M4Eo can be difficult. Characterization of the two genes involved in the inv(16)(p13q22), CBF beta and MYH11, has allowed the detection of fusion transcripts by reverse-transcriptase polymerase chain reaction (RT-PCR). We have analyzed CBF beta-MYH11 fusion transcripts by RT-PCR in myelomonocytic leukemias, with or without eosinophilia, to determine whether their presence correlates with morphology. Fifty-three cases (11 AML M4Eo; 1 AML M4 with atypical abnormal eosinophils (AML M4 “Eo”); 29 AML M4; 8 AML M5; 3 CMML; and 1 AML M2 with eosinophilia) were analyzed. All 11 typical AML M4Eo were CBF beta-MYH11 positive. The single case of AML M4 with distinctive eosinophil abnormalities was negative by karyotype, RT-PCR and fluorescent in situ hybridization (FISH). Three of 29 (10%) AML M4 without abnormal eosinophils were CBF beta-MYH11 positive, 1 of which did not show any apparent chromosome 16 abnormalities by classical metaphase analysis (2 not tested). Both cases tested also showed MYH11 genomic rearrangement. None of the other leukemias were RT-PCR positive. Follow-up of three patient showed residual positivity in apparent complete remission. These data show that CBF beta-MYH11 fusion transcripts occur not only in the vast majority of typical AML M4Eo, but also in approximately 10% of AML M4 without eosinophilic abnormalities, a much higher incidence than the sporadic reports of chromosome 16 abnormalities in AML M4 would suggest. Taken together with the detection of CBF beta-MYH11 transcripts in the absence of apparent chromosome 16 abnormalities by classical banding techniques, these data show that additional screening by either RT-PCR or FISH should be performed in all AML M4, regardless of morphologic features, to allow accurate evaluation of the prognostic importance of this fusion transcript.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

A new sex dimorphism in the Harderian gland of the frog Rana esculenta

2007 ◽  
Vol 85 (8) ◽  
pp. 909-915 ◽  
Author(s):  
Ismene Serino ◽  
Gaia Izzo ◽  
Diana Ferrara ◽  
Michela d’Istria ◽  
Sergio Minucci
Keyword(s):  
Sexual Dimorphism ◽  
Secretory Activity ◽  
Harderian Gland ◽  
Polymerase Chain Reaction Analysis ◽  
Rana Esculenta ◽  
Rt Pcr ◽  
Polymerase Chain ◽  
Two Peaks ◽  
Constant Expression

The Harderian gland (Hg), the only gland found in the orbit of the frog Rana esculenta L., 1758, probably plays a role in orbital lubrication. The secretory activity of the Hg is seasonal, showing the highest activity in summer. There is little information on Hg gene expression; previously, we identified a mRNA named harderin, whose deduced protein has no homology with other proteins. Differential expression of the harderin transcript between the sexes expressed during the annual cycle implies sexual dimorphism. RT–PCR (reverse transcription – polymerase chain reaction) analysis, revealed that harderin is expressed during the entire year in the Hg of both sexes. It shows a higher level of expression in the female glands than that of male glands. Two peaks of expression, in February and in June, were observed in the female glands, while only the February peak was observed in those of males. These observations were supported by in situ hybridization. Experiments involving gonadectomy and (or) hormonal replacement therapy showed a significant decrease in harderin in the Hg of females; this effect is prevented by estradiol (testosterone had no effect), while ICI (antiestrogen) counteracts the hormonal prevention, suggesting that this sexual dimorphism is under estradiol control. The constant expression of harderin mRNA during the year suggests a probable constitutive role for this molecule.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

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