Protein fragment complementation assay (PCA) is based on the interaction between two protein partners (e.g. target antigen and antibody), which are genetically fused to the two halves of a dissected marker protein. Binding of the two partners reassembles the marker protein and hence reconstitutes its activity. In this work we have developed the first application of beta-lactamase-based PCA for the isolation of single chain Fv fragments (scFvs) binding to the human receptor RON from a naïve library. Specific scFvs with the ability to immunoprecipitate could be isolated after a single round of PCA selection from an scFv repertoire previously pre-selected by phage display. Furthermore, the PCA was used to successfully map the epitopes recognized by the selected scFvs by screening them against a small library of random RON fragments.
The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10−3 for bcr1 and bcr3 and 10−2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL.
Until today, JAK2 alterations have been mainly associated with myeloid malignancies among which they play a key pathogenic role in chronic myeloproliferative neoplasms. More recently, aberrations involving the JAK2 gene have also been reported in lymphoid diseases, including acute leukemia and lymphomas. In addition, the constitutively activating JAK2V617F mutation has been identified in some patients affected by B-chronic lymphocytic leukemia with a concomitant myeloproliferative neoplasm. Interestingly, these cases could help us to better understand the pathogenesis of these myeloid and lymphoid diseases and reveal if they share a common ancestral progenitor or just coincide. The involvement of JAK2 in lymphoid neoplasms may suggest the possibility of new therapeutic approaches broadening the use of JAK1-2 inhibitors also to these malignancies.
2515 Background: The molecular identification of the BCRABL transcripts in clinical samples is actually based on a conventional Reverse Transcription Polymerase Chain reaction (RT-PCR). Here we present a novel molecular method, based on Loop Mediated Amplification assay that, starting from RNA (RT-LAMP) in one single tube reaction ensures a rapid and simultaneous detection of either the BCR-ABL p190 or p210 fusion transcript as well as the housekeeping gene used as internal quality control. Methods: The BCR-ABL RT-LAMP is a multiplex, isothermal method for retro-transcription, amplification and detection of the Minor (p190) and Major (p210) t(9;22) transcripts and the endogenous Gusb RNA, as internal control for validation of negative results. The employment of fluorescent specific probes allows real-time monitoring of the reaction, so that the test result is obtained in a single, homogeneous step. RT-LAMP is carried out on the Liaison IAM, a 8-wells manageable instrument suitable for isothermal reactions. Liaison IAM incubates at constant temperature, monitors the fluorescent signals and the data produced can be analyzed, upon connection to up to 6 other instruments, for a throughput of 8 to 48 samples. Thanks to the three channels of fluorescence, it can monitor multiplex assays, providing elaborated final objective results with no need of further data analysis by the operator. Results: The level of sensitivity of the triplex BCR-ABL RT-LAMP has been analytically evaluated directly on serial dilutions of RNA extracted respectively from t(9;22) positive cell lines (TOM-1 for p190 or K-562 for p210) into wild type RNA from HL-60 cell line (30 replicates). The p190 and p210 transcripts have been detected and distinguished down to 10−4 and 10−5respectively within 50 minutes. The assay demonstrated 100% specificity since 70 replicates of wild type RNA from 7 cell lines resulted BCR-ABL negative and GUSb positive (internal amplification control). This assay was validated on 60 clinical samples (30 white blood cells RNA from Chronic Myeloid Leukemia, 30 mononuclear cells RNA from B-lineage Acute Lymphoblastic Leukemia). All these samples were obtained at diagnosis and were previously analyzed by conventional RT-PCR. RT-LAMP detected and identified the BCR-ABL fusion transcripts correctly in all cases with a 100% concordance with the reference method. Fully concordant results were obtained also on 30 RNA samples from patients affected by t(9;22) negative hematologic malignancies and on 30 RNA samples obtained from healthy donors in which the RT-LAMP amplified exclusively the housekeeping GUSb transcript. Conclusions: The triplex p190-p210-GUSb RT-LAMP is a one-step procedure for specific, highly sensitive and rapid molecular detection of the BCR-ABL fusion transcripts. The semi-automatic instrument Liaison IAM, simplifies the entire procedure, reduces the contamination risks deriving from the conventional, multi step RT-PCR and significantly improve the diagnostic lab routine. Disclosures: D'agostini: DIASORIN S.p.A: Employment. Minnucci:Diasorin S.p.A.: Employment. Amicarelli:DIASPRIN S.p.A.: Employment. Pultrone:DIASORIN S.p.A.: Employment. Tettamanzi:DIASORIN S.p.A.: Employment. Salmoiraghi:DIASORIN S.p.A.: Consultancy. Spinelli:DIASORIN S.p.A: Consultancy. Colotta:DIASORIN S.p.A: Employment. Rambaldi:DIASORIN S.p.A: Consultancy.
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