MRD Monitoring in AML Patients with MLL-MLLT4 by Quantification of Fusion Gene Transcripts and Genomic Chromosomal Breakpoint Sequences

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4888-4888
Author(s):  
Grigory Tsaur ◽  
Anna Ivanova ◽  
Olga Plekhanova ◽  
Tatyana Riger ◽  
Yulia Yakovleva ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Fusion Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Exon 2 ◽  
Standard Curve ◽  
Healthy Donors ◽  
Intron 1 ◽  
Exon 9 ◽  
Chromosomal Breakpoint

Abstract Statement. The MLL-MLLT4 (former MLL-AF6) fusion gene (FG) is relatively rare genetic abnormality, predominantly found in AML. It averages 3–5% among other MLL rearrangements. Here we present data of MRD monitoring in 2 patients with AML carrying an MLL-MLLT4 rearrangement by 2 approaches: using FG transcript at RNA/cDNA level in comparison with FG at genomic DNA level. Materials and methods. Patients (pts) were diagnosed according French-American-Britain (FAB) classification. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics, FISH and reverse transcriptase PCR (RT-PCR). RT-PCR products were directly sequenced afterwards. In both cases identification of genomic chromosomal breakpoint sequences within MLL and MLLT4 genes was done by long-distance inverse PCR (LDI-PCR). MRD quantification in genomic DNA was performed using patient-specific primers and probes by real-time quantitative PCR (RQ-PCR). 500 ng of DNA was used per reaction. Standard curve was received by serial 10-fold dilutions of pts’ DNA into the DNA isolated from pooled lymphocytes of ten healthy donors. b-actin was used as DNA quality and quantity control. Detection of FG transcript kinetics during treatment was performed by RQ-PCR according to “Europe Against Cancer” recommendations for normalization by using control gene ABL (Gabert J. et al Leukemia, 2003, 17) and for using 10-fold dilutions of plasmids carrying MLL-MLLT4 fragment as source of standard curve. MRD value for cDNA targets were estimated as previously described (Beillard E. et al Leukemia, 2003, 17). Each sample was run in triplicates. According to the treatment design, time-points for MRD estimation were scheduled before each block of treatment. Totally 5 samples were evaluated in each patient (initial and 4 follow-ups). FLT3-ITD status was estimated at the time of diagnosis. Informed consent was obtained in both cases. Patients’ characteristics, treatment and clinical outcome Case # 1 Case #2 Age 58 13 Sex Male Male Initial WBC*106/ml 5.5 94.9 Immunophenotype CD34+CD117+HLA-DR+ CD11c+CD13+CD33+CD65+ CD34+CD117+CD13+ CD33+CD45+MPO+ Cytogenetics 46, XY, del(5)(q?), der(5)t(5;6;11) (q22;q15q27;q23), der(6)t(5;6) (q22;q15), del der(11) 46, XY, t(6;11)(q27;q23) FISH with LSI MLL MLL deletion MLL split RT-PCR MLL-MLLT4 positive MLL-MLLT4 positive MLL-MLLT4 FG transcript exon 9-exon 2 exon 9-exon 2 Localization of genomic chromosomal breakpoint within MLL and MLLT4 intron 9-intron 1 intron 9-intron 1 FLT3-ITD Negative Negative Induction treatment 7+3 AIE Consolidation therapy 2× HAM 2× HDAC 1× HAM 1× FLAG 1× HAE Maintenance − + Duration of therapy, months 8 7 Achievement of CR + + OS, months 8 7 EFS, months 6 5 Current status Alive in CR Alive in CR Results. Despite of achievement of CR, MLL-MLLT4 FG transcripts were detected in every sample tested after induction and consolidation chemotherapy by RQ-PCR. MRD value in case #1 in cDNA was fluctuated significantly within 2 log. Although in case #2 there was successive reduction from 260% at the beginning of treatment till 0.7% before maintenance therapy (after HAE block). Limited dilution series of a MLL-MLLT4-positive RNA into RNA of ten healthy donors showed a sensitivity limitation of 1E–05. For quantification of genomic chromosomal breakpoint sequences b-actin was amplified in each well. Deviation between Ct values of b-actin in different wells did not exceed ±2.0. In case # 1 the standard curve of the RQ-PCR assay for MLL-MLLT4 FG had slope of −3.19. Correlation coefficient was 0.987. Quantitative range of this assay was 1E–04 and sensitivity 1E–5. It was also observed a considerable variation of MRD levels in genomic DNA during treatment, like it was observed in MRD monitoring by FG transcripts. Fluctuations run up to 2.5 log. In case #2 the standard curve of the RQ-PCR assay for MLL-MLLT4 had a slope of −3.63 with correlation coefficient 0.992. Quantitative range of this assay was reached 1E–04 with sensitivity 1E–05. MRD level in this patient constantly decreased. Conclusions. The same tendency has been shown in each patient: fluctuations of MRD levels (2.5 log in case #1) and successive reduction (case #2). Results received at RNA/cDNA level and in genomic DNA cannot substitute each other, but they can be used as additives. It has been demonstrated that quantification of MLL-MLLT4 FG in genomic DNA is precise and suitable for MRD monitoring.

scholarly journals Exon-skipping in BCR/ABL is induced by ABL exon 2

2000 ◽  
Vol 348 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Brian D. LICHTY ◽  
Suzanne KAMEL-REID
Keyword(s):  
Alternative Splicing ◽  
Genomic Dna ◽  
Chronic Myelogenous Leukaemia ◽  
Fusion Gene ◽  
Exon Skipping ◽  
Cell Types ◽  
Myelogenous Leukaemia ◽  
Sequence Element ◽  
Exon 2 ◽  
Alternatively Spliced

The BCR/ABL fusion gene is pathognomonic for chronic myelogenous leukaemia (CML). We have previously reported alternative splicing of BCR/ABL, as indicated by the detection of both p190- and p210-encoding transcripts, in about 60% of CML patient samples. These exon-skipping events involved the joining of ABL exon 2 to variable upstream BCR exons. Similarly, ABL exon 2 is alternatively spliced to either of two upstream ABL exons (1a or 1b) in c-ABL. We have constructed BCR and BCR/ABL minigenes to study this phenomenon in more detail. These constructs were transfected into various cell types and splicing was assessed by reverse transcriptase PCR. Whereas the basic BCR minigene expressed exon-inclusive transcripts only, insertion of genomic DNA spanning ABL exon 2 induced exon-skipping but only when expressed in the CML cell lines K562 and EM3. In this study we localized the required sequence element to ABL exon 2 itself. These results mimic the splicing phenotype displayed by most CML patients. We propose a model where a trans-factor present in some CML cells interacts with ABL exon 2 pre-mRNA to promote skipping of upstream BCR exons.

scholarly journals Molecular basis of RhD-positive/D-negative chimerism in two patients

2004 ◽  
Vol 10 (1-2) ◽  
pp. 228-241
Author(s):  
S. S. Eid
Keyword(s):  
Molecular Basis ◽  
Genomic Dna ◽  
Major Change ◽  
Base Change ◽  
Cell Populations ◽  
Rt Pcr ◽  
Single Base ◽  
Intron Boundary ◽  
Single Base Change ◽  
Exon 9

This study investigated two patients with Rh chimerism:patient A, a healthy individual, and patient B with myelofibrosis. Flow cytometry studies showed two red blood cell populations of Rh phenotypes R1r and rr at percentages of about 25% and 75% respectively. Normal RhD transcript sequences were found following RT-PCR. Genomic DNA [gDNA] showed normal exon, intron, GATA regions and exon/intron boundary sequences except for a single base change in intron 7 [C –>A] of exon 7 in patient A. The major change found in both patients was the absence of RHD exon 9 DNA in gDNA isolated from peripheral blood. These findings suggest a somatic mutation, probably in a stem cell common to the myeloid lineage of both patients, and indicate that patient A may undergo malignant transformation in the future

scholarly journals Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays

1998 ◽  
Vol 36 (3) ◽  
pp. 628-633 ◽  
Author(s):  
Gerd Haberhausen ◽  
Judith Pinsl ◽  
Carl-Christoph Kuhn ◽  
Christine Markert-Hahn
Keyword(s):  
Reverse Transcription ◽  
Dynamic Range ◽  
Small Region ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Standard Curve ◽  
Reverse Transcription Pcr ◽  
Parallel Data ◽  
Rna Fragments

Four different standardization approaches based on a competitive reverse transcription (RT)-PCR assay were compared with a noncompetitive assay based on an external standard curve. Criteria for assessment were accuracy in quantitation, correctness of recovery, sensitivity, dynamic range, reproducibility, throughput, and convenience of sample handling. As a model system, we used the 5′-noncoding region of hepatitis C virus (HCV) for amplification in all quantitative RT-PCRs. A computer program that allowed parallel data processing was developed. Surprisingly, all methods were found suitable for accurate quantitation and comparable with respect to the criterion correctness of recovery. All results differed only by a factor of about 2. The reason for this finding might be that all of our mimics, as well as the wild-type genome of HCV, exhibited exactly the same amplification and hybridization efficacy. Moreover, minimal competition occurred in our experiments over a 5-log dynamic range. A further topic of our investigation was the comparison of two different competitive RNA fragments, mimics, with regard to their suitability as internal standards. One was a heterologous mimic, in which only the primer binding sites were identical to the wild type. The second one was a homologous mimic identical to the wild type except for a small region used for differential hybridization, which was replaced by a permutated sequence of the same length. Both the homologous and heterologous internal mimics were found appropriate for an accurate competitive RT-PCR assay, provided that amplification efficacy, as well as capture efficacy, is proven identical for both analyte and mimic.

A Novel FOXP1-PDGFRA Fusion Gene in A Case of Chronic Eosinophilic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2830-2830
Author(s):  
Yuka Sugimoto ◽  
Akiko Sada ◽  
Fumihiko Monma ◽  
Kohshi Ohishi ◽  
Masahiro Masuya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Genomic Dna ◽  
Low Dose ◽  
Fusion Gene ◽  
Fish Analysis ◽  
Fusion Partner ◽  
Rt Pcr ◽  
Total Rna

Abstract Abstract 2830 Introduction Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 is a new major category in the 2008 WHO classification of myeloid malignancies. FIP1L1-PDGFRA fusion gene is currently the most common abnormality in this category, but there are some other fusion genes incorporating part of PDGFRA. In a case of myeloproliferative neoplasms (MPN) with eosinophilia and hepatosplenomegaly, karyotype by G-banding and fluorescence in situ hybridization (FISH) for 4q12 rearrangements indicated a PDGFRA rearrangement other than FIP1L1-PDGFRA, and a novel FOXP1-PDGFRA fusion gene was identified. Case presentation A 44-year-old male visited a clinic because of wet cough for one year. His peripheral blood showed leukocytosis of 43.15 × 109 /L with eosinophilia up to 57.5%, mild erythrocytosis (Hb 17.3 g/dL), and thrombocytopenia of 86 × 109 /L. CT scan of the abdomen revealed hepatosplenomegaly. He was referred to our hospital and received oral PSL (1 mg/kg) first, because pulmonary eosinophilic infiltration was suspected by follow-up CT findings. Pulmonary infiltration and his cough disappeared rapidly in a week, but his leukocytosis with eosinophilia was exacerbated again with PSL tapering. His bone marrow at the time of admission disclosed hypercellular marrow with myeloid hyperplasia and eosinophilia, of which karyotype was 46, XY, t(3:4)(p13;q12), inv(9)(p12q13) in all of 20 metaphases. FISH analysis with tricolor 4q12 rearrangement probe set indicated that PDGFRA was disrupted in 97.3% of his peripheral blood cells. These cytogenetic abnormalities of his bone marrow cells suggested involvement of PDGFRA fusion gene except for FIP1L1-PDGFRA and did not disappear after steroid administration for 2 weeks. After low-dose of imatinib (100 mg/day) was started, he achieved a hematological response within 5 days, and PSL could be gradually tapered off. 3 months after therapy, he obtained complete cytogenetic response (CCyR). He has been in CCyR and free of symptoms for more than 6 months with only low-dose imatinib. Methods and Results Genomic DNA and total RNA were isolated from white blood cells in his peripheral blood at diagnosis. Complementary DNA was synthesized from total RNA. FIP1L1-PDGFRA fusion transcript was proved to be negative by RT-PCR. Molecular cloning with 5′-RACE-PCR revealed a novel mRNA in-frame fusion between exon 23 of FOXP1 and a truncated PDGFRA exon12. Reciprocal PDGFRA-FOXP1 transcripts were confirmed by RT-PCR analysis and FOXP1-PDGFRA genomic DNA sequence was determined with genomic PCR. As in the case with FIP1L1-PDGFRA, the breakpoint of PDGFRA in FOXP1-PDGFRA was located between the two tryptophan (W) residues of the putative WW-domain. Meanwhile, the other breakpoint was near inverted repeat in intron 23 of FOXP1, which is presumed to be very fragile site. By FISH analysis after magnetic cell sorting with MicroBeads, the 4q12 abnormality attributed to FOXP1-PDGFRA fusion gene was detected in granulocytes, but not in CD19-positive B or CD3-positive T cells. Discussion In a case with chronic eosinophilia harboring 46, XY, t(3:4)(p13;q12), inv(9)(p12q13), novel FOXP1-PDGFRA fusion gene was identified. Similar karyotypic abnormality harboring t(3:4)(p13;q12) was reported in a case of MPN with chronic eosinophilia, but responsible fusion gene was not identified (Myint H, et al. Br J Haematol. 1995). FOXP1 is a transcription factor which is implicated in a variety of cellular processes and has a role in immune regulation and carcinogenesis (Wlodarska I, et al. Leukemia. 2005). As a fusion partner of FOXP1, PAX5 and ABL1 are reported in cases with acute lymphoblastic leukemia. Thus, this is a first report showing that FOXP1-PDGFRA fusion gene is involved in hematologic malignancy. It is likely that FOXP1-PDGFRA is constitutively activated tyrosine kinase, which does not depend on dimerization but on the disruption of an autoinhibitory juxtamembrane domain encoded by exon 12 of PDGFRA from its structure. Eosinophilia responded well to low dose of imatinib as observed in CEL with FIP1L1-PDGFRA. Conclusion FOXP1-PDGFRA was identified in CEL for the first time. This is the eighth reported fusion gene associated with PDGFRA in CEL so far. Our patient with FOXP1-PDGFRA promptly responded to low-dose of imatinib as same as other cases with PDGFRA abnormalities. Further investigation is still in progress. Disclosures: No relevant conflicts of interest to declare.

Identification and Functional Significance of Novel Type of Structurally Aberrant Transcripts of DCC In B-Cell Malignancies.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3623-3623
Author(s):  
Hisao Nagoshi ◽  
Tomohiko Taki ◽  
Junya Kuroda ◽  
Kazuhiro Nishida ◽  
Minako Gotoh ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Line ◽  
Cell Lines ◽  
Western Blotting ◽  
Rt Pcr ◽  
Exon 2 ◽  
Intron 1 ◽  
Exon 1 ◽  
Dcc Gene ◽  
Tumor Suppressor Gene Inactivation

Abstract Abstract 3623 Many genetic alterations have been detected in various tumors, and their expression levels have also been investigated concerning cancer development or progression. Gene expressions have so far been examined by Northern blotting, reverse-transcriptase-polymerase chain reaction (RT-PCR), microarray and real-time PCR. There are several papers reporting that the expression level of genes, such as EVI1 and BAALC, are associated with clinical outcome in some types of hematologic malignancies, however, we do not know whether the genes detected are structurally normal or abnormal, because the point mutation, fusion gene and alternative splicing could not be detected by conventional method without sequencing. We previously found recurrent genomic copy number variations at 18q21–22 in malignant lymphoma (ML) and multiple myeloma (MM) cell lines by oligonucleotide array analysis. When we analyzed the several cancer-related genes a t 18q21–22, strange expressions of DCC gene were found in some MM cell lines by chance. The expression of DCC gene were analyzed in 21 MM and 11 ML cell lines by RT-PCR using 15 primer pairs covering the full length of DCC, and we found 4 MM cell lines which did not express the transcripts containing exon 1 but those containing other exons. This result made us predict the abnormal form of DCC transcripts fused to unknown sequences instead of exon 1, therefore, we tried to clone them using cDNA bubble PCR method. Some products were obtained and sequence analysis revealed that the 2 kinds of bubble PCR products contained unknown sequences fused to exon 2 of DCC. BLAST search revealed that both unknown sequences were different parts of intron 1 of DCC, which are not connected to exon 2 normally. Sequential analysis of DCC expression by RT-PCR revealed that both normal (containing exon 1) and abnormal (lacking exon 1 and containing intron 1) transcripts were detected in 16/21 (76.2%) MM cell lines, 8/33 (24.2%) MM cases, and 0/11 (0%) ML cases, and that only abnormal transcripts were in 4 (19.0%) MM cell lines, 10 (30.3%) MM cases, and 5 (45.5%) ML cases. Furthermore, another abnormal DCC transcripts which lacked both exon 1 and intron 1 were detected in 12 (36.4%) MM cases, 1 (9.1%) ML cell line, and 4 (36.4%) ML cases. In total, abnormal DCC transcripts were detected in 20 (95%) MM cell lines, 30 (90.9%) MM cases, 1 (9.1%) ML cell line, and 9 (81.8%) ML cases. One (4.8%) MM cell line, 3 (9.1%) MM cases, 10 (90.9%) ML cell lines, and 2 (18.2%) ML cases did not express normal or abnormal transcripts. We next performed Western blotting to detect DCC protein. After immunoprecipitation with the anti-C-terminus of DCC antibody, the DCC protein was detected in 2 MM cell lines with abnormal transcript of DCC, which also expressed normal of DCC clearly. On the other hand, no DCC protein was detected in MM cell lines with abnormal DCC transcripts, which expressed normal DCC transcript weakly. These findings suggest that DCC protein detected by Western blotting is derived from the normal transcript, and that abnormal DCC transcripts result in the defect of their function. DCC, which encodes a receptor for netrin-1, would control apoptosis and be concerned in oncogenesis. Loss of one allele or decreased expression of DCC has been reported in many malignancies including solid tumors, leukemia and ML. It has been considered that DCC inactivation by these mechanisms constitutes a critical event in the development of these tumors. In this study, although partial expression of DCC could be detected by RT-PCR in many MM and ML cases, most of them were abnormal DCC, which would result in lacking DCC function. In addition, our data indicated that the creation of aberrant transcript by possibly abnormal alternative splicing may be another mechanism of tumor suppressor gene inactivation, therefore, analysis of gene expression should be performed in view of the genomic structure of genes. Disclosures: No relevant conflicts of interest to declare.

Identification of the Novel Chimeric Gene, PVT1-WWOX, in Multiple Myeloma with 8q24 Abnormality

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1823-1823
Author(s):  
Hisao Nagoshi ◽  
Tomohiko Taki ◽  
Kazuhiro Nishida ◽  
Junya Kuroda ◽  
Yoshiaki Chinen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Cell Line ◽  
Copy Number ◽  
Copy Number Change ◽  
Suppressor Gene ◽  
Oligonucleotide Array ◽  
Rt Pcr ◽  
Intron 1 ◽  
Exon 9

Abstract Abstract 1823 Genetic abnormalities play a crucial role in the pathogenesis of various malignancies, including multiple myeloma (MM). Secondary cytogenetic abnormalities implicated in MM progression include 8q24 rearrangements, gain of the long arm of chromosome 1 (1q+), and loss of the short arm of chromosome 17 (17p-). The 8q24 rearrangements, including MYC and PVT1, have been identified by conventional cytogenetic analysis in 3.5–5.0% of MM patients and by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) in 9.5–20%. 8q24 rearrangements are frequently associated with advanced disease in MM patients and MM cell lines. Ig chromosomal translocations, such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% of MMs with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14 and 16q22, in which no candidate genes have been delineated so far, have also been identified as translocation partners. MYC has long been a possible candidate target gene for 8q24 rearrangements; however, many of the breakpoints within 8q24 have been assigned to various regions that encompassed more than 2 Mb centromeric or telomeric to MYC. We have previously found frequent PVT1 rearrangements in MM. PVT1 rearrangements were detected in 7 of 12 patients (58.3%) and in 5 of 8 cell lines (62.5%) with 8q24 abnormalities. A combination of SKY, FISH, and oligonucleotide array identified several partner loci of PVT1 rearrangements, such as 4p16, 4q13, 13q13, 14q32 and 16q23–24, and identified a chimeric gene, PVT1 - NBEA, resulting in high expression of abnormal NBEA in a cell line with t(8;13)(q24;q13), AMU-MM1, suggesting PVT1 rearrangements play significant roles in myelomagenesis. In this study, we analyzed RPMI8226 cell line in detail to identify other partner genes of PVT1 in these partner loci. SKY analysis revealed a complex karyotype including der(16)t(16;22)ins(16;8)(q23;q24) in this cell line. Oligonucleotide array analysis clearly demonstrated that the copy number change at 8q24 occurred within intron 1 of PVT1, and at 16q23, the copy number change occurred within intron 8 of WWOX, indicating that the translocation breakpoints of 8q24 and 16q23 were within intron 1 of PVT1 and intron 8 of WWOX, respectively. Based on these results, RT-PCR analysis was performed to detect chimeric products and direct sequencing of this product revealed the fusion of 5'-PVT1 exon 1 with WWOX exon 9-3'. The expression level of WWOX exon 9 was higher than WWOX exon 8–9 detected by semi-quantitative RT-PCR in RPMI8226, suggesting that high expression of WWOX derived from PVT1 - WWOX chimeric transcript, like PVT1 - NBEA. WWOX is generally considered to be a candidate tumor suppressor gene, and known to have a proapoptotic effect by participating in the tumor necrosis factor (TNF) apoptotic pathway and via direct physical interaction with p53 and its homolog p73. However, immunohistochemistry revealed that WWOX protein level were rather elevated in gastric and breast carcinoma. Therefore, WWOX seemed not to act as tumor suppressor gene simply. Although both NBEA and WWOX are located at common fragile site, usually contributing to gene inactivation, FRA13A and FRA16D respectively, these genes highly express via fusion to PVT1. These findings indicate that PVT1 rearrangements play significant roles in myelomagenesis via translocation and fusion to cancer-related genes. Disclosures: No relevant conflicts of interest to declare.

Update on the large-scale screening of ALK fusion oncogene transcripts in archival NSCLC tumor specimens using multiplexed RT-PCR assays.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7594-7594 ◽  
Author(s):  
Tianhong Li ◽  
Eric Huang ◽  
Sonal Desai ◽  
Laurel Beckett ◽  
Craig Stephens ◽  
...  
Keyword(s):  
Large Scale ◽  
Fusion Gene ◽  
Standard Of Care ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts ◽  
Gene Transcripts ◽  
Pcr Assays ◽  
Alk Positive ◽  
Large Scale Screening

7594 Background: The ALK inhibitor crizotinib offers a new standard of care for advanced NSCLC patients with EML4-ALK fusion oncogenes. We previously reported a 4.0% frequency of EML4-ALK fusion oncogene transcripts detected in 1889 NSCLC specimens in the RGI database (Li et al., ASCO 2011). Methods: Patented single and multiplexed RT-PCR assays suitable for rapid and accurate detection of all variants of ALK fusion oncogene transcripts were used as previously described, including all 9 known EML4-ALK fusion gene transcripts and ALK RNA levels (Danenberg, ASCO 2010). The sensitivity and specificity on archival formalin-fixed, paraffin-embedded tumor specimens are 99% and 100%, respectively. We here update the detection of EML4-ALK fusion transcripts in the RGI database. Results: Between 12/2009 and 09/2011, 4750 NSCLC specimens in the RGI database were tested for the presence of ALK fusion transcripts. We found 152 (3.2%) NSCLC cases with EML4-ALK fusion positivity, including 87 (57.2%) V1, 15 (9.9%) V2, 47 (30.9%) V3, and 3 (2.0%) V5a variants. Median age (range): 61.1 (33-96). Female: 74 (49%). All EML4-ALK-positive tumors were adenocarcinomas. No EGFR or K-Ras mutation was detected in ALK fusion-positive samples. Expression of chemotherapy-related biomarkers was available from 63 (female: 31, 49%) EML4-ALK-positive cases in the database: 43 (68%) had low TS level of <2.33; 40 (63.5%) had low ERCC1 level of <1.7, and 25 (40%) had low RRM1 level of <0.97. Conclusions: This RT-PCR assay provides a tool for rapid, large-scale screening of NSCLC FFPE tissues for EML4-ALK fusion gene transcripts. The relative value of this RT-PCR assay as a companion diagnostic test for drugs targeting ALK merits evaluation in comparison with the FDA approved ALK FISH test.

scholarly journals ANGI-05. ORIGIN OF MICROVASCULAR PROLIFERATION IN PILOCYTIC ASTROCYTOMA - MVP IN PILOCYTIC ASTROCYTOMA MIGHT PARTIALLY COMPRISE TUMOR-DERIVED CELLS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi31-vi31
Author(s):  
Shinji Yamashita ◽  
Hideo Takeshima ◽  
Takashi Watanabe ◽  
Kiyotaka Yokogami
Keyword(s):  
Tumor Cells ◽  
Pilocytic Astrocytoma ◽  
Fusion Gene ◽  
Digital Pcr ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Microvascular Proliferation ◽  
Braf Fusion ◽  
Cellular Components

Abstract Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVP in PA contained tumor-derived cells. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was characterized by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP may be tumor-derived.

scholarly journals Dengue IgG Seropositivity and Zika Viral Load

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S301-S302
Author(s):  
Jennifer Read ◽  
Luisa I Alvarado ◽  
Brenda Torres-Velasquez ◽  
Jorge L Munoz-Jordan ◽  
Manuela Beltran ◽  
...  
Keyword(s):  
Puerto Rico ◽  
Linear Regression ◽  
Febrile Illness ◽  
Denv Infection ◽  
Wilcoxon Test ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Zikv Infection ◽  
Chi Square ◽  
Standard Curve

Abstract Background Secondary dengue virus (DENV) infections are typically more severe than primary infections. It is not known whether previous DENV infection is associated with higher Zika virus (ZIKV) quantitative RT-PCR results (viral loads (VLs)) in areas endemic for DENV such as Puerto Rico. Our objective was to analyze the association between previous DENV infection (DENV IgG-positive) and ZIKV VL among children with symptomatic ZIKV infection enrolled in the Sentinel Enhanced Dengue and Acute Febrile Illness Surveillance System (SEDSS) in Puerto Rico. Methods The study population for this analysis comprised individuals &lt;18 years of age enrolled in SEDSS during 2016 who were ZIKV PCR-positive in serum (using the CDC Trioplex RT-polymerase chain reaction (RT-PCR) assay) within 5 days post-onset (DPO) of symptoms. ZIKV VLs (genome copies/mL) were determined using an RNA standard curve generated from the RT-PCR assay target amplicons. An in-house ELISA was used to ascertain the presence or absence of serum DENV IgG. Trends were assessed using Jonckheere-Terpstra and Chi-square for proportions tests. The Mann–Whitney-Wilcoxon test was used to compare medians. Linear regression modeling was used to determine the association between DENV IgG and ZIKV VL. Results Of the 319 individuals who met inclusion criteria, 163 have dengue IgG assays completed to date. Of these, 90/163 (55%) were DENV IgG-positive and 73/163 (45%) were DENV IgG-negative, and did not vary by sex (P = 1.00). However, the proportion of patients with DENV IgG-positivity increased with age (P &lt; 0.001) (Figure). Overall, the median (interquartile range, IQR) ZIKV VL was 23,110 (7,452–84,003), and did not vary by age (P = 0.11) or sex (P = 0.33). However, the median ZIKV VL varied by DPO: 26,230 (DPO&lt;3; n = 117), 15,159 (DPO≥ 3; n = 46), P = 0.002. The median (IQR) ZIKV VLs were: 24,073 (10,938–73,130) in DENV IgG-negative specimens and 22,658 (7,332–89,322) in DENV IgG-positive specimens (P = 0.91). Linear regression indicated no association between DENV IgG and ZIKV VL (P = 0.54). Conclusion DENV IgG-positivity increased with age among children with symptomatic ZIKV infection. ZIKV VLs did not vary by age, but decreased with increasing DPO. There was no association between DENV IgG and ZIKV VL. Disclosures All authors: No reported disclosures.

scholarly journals Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus

2021 ◽  
Vol 8 ◽  
Author(s):  
Yi-Lun Xie ◽  
Dian-Hong Lv ◽  
Xiao-Hui Wen ◽  
Qi Zhai ◽  
Man-Lin Luo ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Foot And Mouth Disease ◽  
Coefficient Of Determination ◽  
Potential Contribution ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Standard Curve ◽  
Mouth Disease Virus ◽  
B Virus

Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease virus and Senecavirus A. The limit of detection of the assay was 4.46 copies per reaction. A standard curve was plotted, with a coefficient of determination of 0.999 in the concentration range of 100-108 copies/μl. The reproducibility of the assay was acceptable, with the standard deviations of cycle threshold values lower than 1.00 in both intra- and inter-assay. Of 200 samples collected from 150 head of cattle in recent years in China, 11% (22/200) of the samples tested positive in the assay, i.e., 4.6% (7/150) of the cattle were BRBV positive. This study provides an efficient diagnostic tool for the epidemiological investigations of BRBV.

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