A validated real-time quantitative PCR approach shows a correlation between tumor burden and successful ex vivo purging in follicular lymphoma patients

Ladetto, Marco; Sametti, Selina; Donovan, John W.; Ferrero, Dario; Astolfi, Monica; Mitterer, Manfred; Ricca, Irene; Drandi, Daniela; Corradini, Paolo; Coser, P; Pileri, Alessandro; Gribben, John G.; Tarella, Corrado

doi:10.1016/s0301-472x(00)00651-2

Cited by 65 publications

(46 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…For example, Hirt and Dolken (3) have demonstrated that quantitative detection of circulating t(14;18) by real-time PCR in follow-up samples of FL patients after autologous bone marrow transplantation predicts clinical course of the disease. Similarly, Ladetto et al (5) have shown that tumor burden in stem cell harvests detected by real-time PCR for t(14;18) can predict the effectiveness of therapeutic intervention in FL patients. These studies demonstrate that real-time PCR is a reliable tool that can be used for monitoring minimal residual disease and in the evaluation of treatment effectiveness in FL patients.…”

Section: Discussionmentioning

confidence: 91%

Real-Time t(14;18)(q32;q21) PCR Assay Combined with High-Resolution Capillary Electrophoresis: A Novel and Rapid Approach that Allows Accurate Quantitation and Size Determination of bcl-2/JH Fusion Sequences

et al. 2002

View full text Add to dashboard Cite

Follicular lymphoma is characterized by the presence of the t(14;18)(q32;q21) chromosomal translocation that juxtaposes the bcl-2 gene at 18q21 with the immunoglobulin heavy chain (IgH) locus at 14q32. We have previously shown that accurate quantitation of t(14;18)-carrying cells in follicular lymphoma patients can be achieved by non-gelbased real-time TaqMan polymerase chain reaction (PCR; Applied Biosystems, Foster City, CA). Since our report, several studies have demonstrated that real-time PCR is highly sensitive and a reliable tool for evaluating treatment effectiveness and for following minimal residual disease in follicular lymphoma patients. Unfortunately, currently available real-time PCR methods do not determine the size of the amplification product, which is useful for excluding contamination and is commonly used as presumptive evidence of clonal identity or disparity when multiple samples from the same patient are analyzed. We describe a modified real-time PCR assay that rapidly allows accurate quantitation and precise determination of the size of the t(14;18) fusion sequence without the need for gel electrophoresis. In this assay, a consensus immunoglobulin heavy chain-joining region gene (JH) primer labeled at its 5' end with the fluorescent dye NED (Applied Biosystems) is included in the real-time PCR assay and thus is incorporated into the bcl-2/JH fusion product. The JH-NED primer did not interfere with the TaqMan probe fluorescent signal or target detection and allowed subsequent amplicon size determination by semiautomated highresolution capillary electrophoresis.KEY WORDS: Follicular lymphoma, High-resolution capillary electrophoresis, Real-time polymerase chain reaction, t(14;18). Mod Pathol 2002;15(4):448 -453Real-time polymerase chain reaction (PCR) for detection and quantitation of bcl-2/JH fusion sequences characteristic of t(14;18) (q32;q21) is being used increasingly to monitor tumor burden and the effect of therapy in patients with follicular lymphoma (FL; 1-5). Real-time PCR methods enable t(14;18) detection without the need to open amplification tubes and are target specific, highly sensitive, and reproducible. Thus, real-time PCR methods have several advantages over conventional PCR techniques that require gel electrophoresis-based amplicon detection and quantitation techniques. Currently available real-time PCR methods to detect the t(14;18) do not allow amplicon size determination. This is a major drawback for a few reasons. First, one cannot easily exclude contamination. Second, size of PCR products from multiple samples (either simultaneous or sequential) is a valuable means of determining clonal relatedness, as the size of bcl-2/JH fusion sequences generally vary from clone to clone (6 -8). Undoubtedly, sequencing of the fusion sequences is the most accepted method for determining clonal relatedness, but sequencing methods can be tedious and time consuming. Third, as t(14;18)-positive cells are detected in normal healthy individuals (9 -12), the ability to quantitate low-level...

show abstract

Section: Discussionmentioning

confidence: 91%

Real-Time t(14;18)(q32;q21) PCR Assay Combined with High-Resolution Capillary Electrophoresis: A Novel and Rapid Approach that Allows Accurate Quantitation and Size Determination of bcl-2/JH Fusion Sequences

et al. 2002

View full text Add to dashboard Cite

show abstract

“…Primers were MBR1int (forward, Table 1) and VLJH (reverse, Table 1); the probe was 5=-6FAM-CTGTTTCAACACAGACCCA-MGBNFQ-3= (Applied Biosystems, modified from Ladetto et al). 16 Real-time PCR reactions were performed in a 15-L total volume containing 3 pmol each primer, 1.5 pmol probe, 2.5 mmol/L MgCl 2 , 0.2 mmol/L each deoxyribonucleotide, 0.5 units AmpliTaq Gold DNA polymerase, and 1.5 L buffer (Applied Biosystems). Samples containing 20 ng DNA were subjected to 45 cycles of denaturation (95°C, 30 seconds), annealing (57°C, 30 seconds), and extension (72°C, 60 seconds).…”

Section: Real-time Pcrmentioning

confidence: 99%

Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations

Lin

Tseng

Rich

et al. 2011

The Journal of Molecular Diagnostics

View full text Add to dashboard Cite

PCR detection of chromosomal translocations and small insertion/deletion mutations is challenging when potential amplicon size varies greatly. Molecular diagnostic laboratories face such difficulties with the BCL2-IGHtranslocation in follicular lymphoma and with internal tandem duplication mutation of the FLT3 gene in leukemia, where breakpoints are widely distributed, mutations may be multiple, signal strength is low, and background noise is elevated. We developed a strategy, called ⌬-PCR, that ensures PCR specificity and identifies individual breakpoints. ⌬-PCR uses two forward primers (external and internal) and a reverse primer simultaneously. The internal primer functions as a probe with a defined distance ⌬ from the external primer. For follicular lymphoma, we prepared upstream, BCL2-specific primers for potential breakpoints to pair with a common, downstream VLJH primer. Multiplexed PCR amplicons are sized by capillary electrophoresis. Each of the upstream pairs has a defined interval separating them that uniquely identifies the breakpoint. The presence of two amplicons with a defined size difference confirms validity of the rearrangement and identity of the specific breakpoint, even if signal strength is low. By testing 40 follicular lymphoma and 12 control specimens from formalin-fixed, paraffinembedded (FFPE) blocks, we showed that multiplex ⌬-PCR is a simple, sensitive strategy to identify translocations with multiple breakpoints or partners. The strategy was also applied to detect minor leukemic clones with internal tandem duplication mutations and could have broader applications for other insertion/deletion and duplication mutations. Chromosomal translocations and small insertion/deletions occur in specific hematological malignancies and solid tumors, and are often definitional genetic events. 1,2Translocations involve breakage and fusion of two or more chromosomes, and insertion/deletions (as considered in the present study) involve breakage within one chromosome. The detection of these chromosomal abnormalities by conventional cytogenetics study, standard Southern blot analysis, fluorescence in situ hybridization (FISH), PCR, and reverse-transcription PCR (RT-PCR) has been used to diagnose specific malignancies and also to monitor minimal residual cancer cells. 3Depending on the specific breakpoint and fusion, chromosomal translocations are classified into two categories.1 Breakage within genes leads to a fusion transcript producing an oncogenic chimeric protein, as in chronic myelogenous leukemia (BCR-ABL1) and synovial sarcoma (SYT-SSX1 or SYT-SSX2). 4 -6 In these cases, diagnostic PCR using genomic DNA is difficult because the potential breakpoint regions in the introns span large distances; however, the fusion transcript can be detected with high sensitivity and specificity by RT-PCR across the involved exon junction at the RNA level. 7,8 The second mechanism of translocation-induced oncogenesis, particularly common in lymphoid malignancies, involves the approximation of an oncogene and an ac...

show abstract

“…Por lo tanto, es importante desarrollar métodos que puedan determinar cuantitativamente los niveles de EMR más que, simplemente, su presencia o ausencia, para establecer rangos asociados a enfermedad. Para ello se esta utilizando la técnica de PCR a tiempo real que es capaz de cuantificar con exactitud el número de copias de un marcador tumoral determinado a partir de una muestra sanguínea del paciente 15 .…”

Section: Características Morfológicas/ Fenotípicasunclassified

Vacuna idiotípica en el tratamiento del linfoma folicular: situación actual y perspectivas futuras

Zabalegui¹,

Cerio²,

Inogés³

et al. 2009

Anales Sis San Navarra

View full text Add to dashboard Cite

RESUMENEl linfoma folicular (LF) está considerado como el segundo tipo de linfoma no-Hodgkin más común, representando más del 20% del total de los linfomas. Es una enfermedad de progresión lenta y curso indolente en la que, a pesar de la buena respuesta al tratamiento, las recaídas son muy frecuentes y cada vez es más difí-cil conseguir respuestas completas. Por ello, se puede considerar que hasta el momento, el LF es incurable. La búsqueda continua de nuevas estrategias terapéu-ticas en enfermedades neoplásicas, junto con un mejor conocimiento del sistema inmunitario, ha llevado a la aparición de una nueva disciplina, conocida con el nombre de inmunoterapia, que aprovecha la capacidad del sistema inmunitario de atacar lo extraño sin dañar lo propio. El LF es un tumor muy apropiado para este tipo de tratamiento por presentar un antígeno específi-co de tumor: el idiotipo de la inmunoglobulina monoclonal expresada en la membrana de todas las células tumorales. Se han realizado diversos estudios en los que se ha probado la inmunoterapia como tratamiento complementario al tratamiento convencional. Recientemente, nuestro grupo ha publicado un estudio en el que se observa claramente que los resultados que se obtienen tras la vacunación idiotípica, cuando se consigue la inmunización adecuada del paciente, son mejores que los obtenidos con quimioterapia sola. En este sentido, es necesario seguir investigando para aclarar si la vacunación idiotípica pudiera no sólo mantener remisiones completas duraderas en los pacientes vacunados, sino incluso conseguir la curación de los mismos. Por ello, resulta interesante abordar un mejor planteamiento de los ensayos clínicos, la mejora de la producción de la vacuna y el estudio de mecanismos de la célula tumoral capaces de modificar la inmunoglobulina específica del tumor.Palabras clave. Linfoma folicular. Inmunoterapia. Vacuna idiotípica. Glicosilación. Splicing alternativo. ABSTRACTFollicular lymphoma is the second most prevalent non-Hodgkin lymphoma, representing 20% of all lymphomas. Follicular lymphoma is an indolent disease with a slow progression in which, although exhibiting a good response to treatment, relapse is very frequent and complete remission is not easy to maintain. Therefore, the disease is regarded as incurable. The search for new therapeutic strategies, together with a better understanding of the immune system, has led to the emergence of a new treatment named immunotherapy. Follicular lymphoma is a malignancy suitable for this kind of treatment given the fact that it is characterized by presenting a unique tumour-specific antigen: the idiotype of the monoclonal immunoglobulin displayed on the membrane of tumour cells. Several studies have been conducted to test immunotherapy as complementary to conventional treatment. In a previous study by our group, a clear benefit was evident is obtained after idiotypic vaccination, when an adequate immunization of the patient is obtained, in comparison to chemotherapy alone. In this sense, analysis is needed of whether idi...

show abstract

A validated real-time quantitative PCR approach shows a correlation between tumor burden and successful ex vivo purging in follicular lymphoma patients

Cited by 65 publications

References 35 publications

Real-Time t(14;18)(q32;q21) PCR Assay Combined with High-Resolution Capillary Electrophoresis: A Novel and Rapid Approach that Allows Accurate Quantitation and Size Determination of bcl-2/JH Fusion Sequences

Real-Time t(14;18)(q32;q21) PCR Assay Combined with High-Resolution Capillary Electrophoresis: A Novel and Rapid Approach that Allows Accurate Quantitation and Size Determination of bcl-2/JH Fusion Sequences

Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations

Vacuna idiotípica en el tratamiento del linfoma folicular: situación actual y perspectivas futuras

Contact Info

Product

Resources

About