The use of comparative duplex PCR in monitoring of patients with non-Hodgkin's lymphoma and chronic lymphocytic leukaemia.

Various quantitative PCR approaches have been utilized during the last years to provide information about the treatment efficacy and the risk of recurrent disease in haematological malignancies. Apart from the frequently used real-time PCR, cost-saving modified standard PCR methods may be applied as well. This report evaluates the utility of the end-point comparative duplex PCR. We have used this method for monitoring of 35 patients with either NHL or CLL and observed a good correlation between quantitative molecular results and clinical outcome. There was also an agreement between comparative duplex PCR and real-time PCR in patients who were monitored by both methods. We therefore believe that use of this technique should be strongly considered instead of simple qualitative detection in monitoring of therapeutic outcome in NHL or CLL patients.

[Clinical importance of semi-quantitative monitoring of lymphomas using the comparative polymerase chain reaction]. / Klinická relevance semikvantitativního monitorování lymfomu uzitím komparativní polymerázové retezové reakce.

BACKGROUND: PCR techniques detecting interchromosomal translocation and clonal immunoglobulin gene rearrangement (IgH) as disease markers in non-Hodgkin's lymphomas (NHL) has been utilised past ten years. However, qualitative PCR detection of persisted minimal residual disease cannot provide clinically useful prognostic information and presently, quantitative approaches are required to predict patient outcome and assess response to the treatment. In some cases, "end-point" quantifying techniques, such as comparative PCR, are applicable and the relative estimation of differences in target quantity may serve in disease monitoring rather than absolute number of target copies. METHODS AND RESULTS: Our method of comparative PCR employs co-amplification of sequences of interest (clonal CDR3, bcl2/Jh) and the segment of Hras 1 gene(ras) as an internal standard. Serial dilutions of stored diagnostic DNAs from blood and bone marrow are examined in the same PCR and, after gel densitometry, the amount of initial target is assessed by comparing exponential products of co-amplification. The comparative PCR assay was utilized in monitoring of NHL patients cured either with conventional therapy, or with high-dose regimens and transplantation with stem cells, or with chimaeric anti-CD20 monoclonal antibody (Rituximab). Results from 50 monitored intervals obtained during several months up to several years were supplemented with clinical statements retrospectively. Some of patients became PCR-negative, reappearance of PCR-positivity was observed as well. The decrease or increase of disease marker corresponded to clinical observations. Results obtained from bone marrow were in agreement with those obtained from blood. CONCLUSIONS: End-point quantifying PCR comparative assay may provide an information on the increased risk of relapse and impact of the therapy. The predictive value of these methods depends on the frequency of sample taking and on the sensitivity of the method, which should be monitored in negative cases.

Optimized multiplex IgH/ras PCR: a tool for quantitative monitoring of B-lymphoproliferative disorders.

The use of quantitative PCR is recommended to monitor the level of residual hematological malignancies. The proposed multiplex IgH/ras PCR uses a co-amplification of the clonal CDR3 rearrangement of the immunoglobulin heavy chain gene (IgH) as a disease marker and a segment of the Hras 1 gene containing codon 61 (ras) as a control gene. Serial dilutions of stored diagnostic DNAs are examined together in the same PCR at a sub-plateau phase and, after analysis by densitometry, the amount of CDR3 product is related to the ras product. An increase of this ratio at comparable amounts of DNA is viewed as an increase of malignant cells. This endpoint PCR quantifying approach appears to be applicable in monitoring B-lymphoproliferative disorders as was shown to be true in B-cell non-Hodgkin's lymphoma and may provide information on disease activity and treatment outcome.

[Detection and significance of chromosome abnormalities in patients with malignant non-Hodgkin's lymphoma using the polymerase chain reaction]. / Detekce a význam chromozomálních abnormalit u nemocných s nehodgkinskými maligními lymfomy metodou polymerázové retezové reakce.

BACKGROUND: Morphology and immunological marker analysis are insufficient to detect neoplastic population in some cases (15%) of non-Hodgkin's lymphomas (B-NHL). Aim of the study was to detect malignancy at molecular level using polymerase chain reaction. METHODS AND RESULTS: We examined a diverse set of B-NHL (90 patients--48 men, range 18-76 years, mean age 53 years and 42 women, range 20-86 years, mean age 54 years) to detect immunoglobulin heavy chain (IgH) rearrangement. 32 patients with centroblastic-centrocytic lymphomas (12 men, range 45-64 years, mean age 52 years and 20 women, range 29-85 years, mean age 53 years) were also studied for translocation (14,18). DNA was isolated from lymphatic nodes, bone marrows and peripheral blood. Translocation (14,18) was founded in 38% lymphatic nodes, 36% bone marrows and in 50% of peripheral blood. The detection rate of IgH PCR varied according to the morphologic type of the analyzed lymphoma specimen. A high detection rate (100%) was observed in low-grade lymphoma, while in high-grade lymphoma was in 62%. In bone marrows samples from follicular lymphomas, IgH PCR positivity was observed in 50% cases without leukaemic blood picture and in 64% cases with lymphoma cells in peripheral blood picture. In peripheral blood with bone marrow infiltration, but without the presence of lymphoma cells (morphological assessment) we observed 71% IgH PCR positive samples. In case, when bone marrow and peripheral blood were morphologic negative, we identified 64% positive cases. Using t(14,18) and IgH PCR we detected neoplastic population in 81% follicular lymphomas. CONCLUSIONS: IgH PCR and t(14.18) PCR are convenient additional technology for detection of neoplastic lymphocytes in B-NHL, particularly when morphology and immunological marker analysis are insufficient.

Monitoring of residual disease in non-Hodgkin's lymphomas by quantitative PCR (preliminary report).

Highly sensitive PCR techniques are often used in molecular monitoring of hematological malignancies, and a quantification of residual disease is important for further prognosis. Here, the limiting dilution methodology and the multiplex IgH/ras PCR are proposed as approaches to molecular monitoring of NHLs. Applying the limiting dilution methodology as a simple dose-response assay for the translocation t(14,18) and CDR3 clonal rearrangement of IgH, critical amounts of total cells determined with stored consecutive diagnostic samples in the same PCR run are compared. Assuming that specific targets are diluted proportionally in dilution of total genomic DNA, the samples showing lower critical concentrations of total DNA are considered as containing higher portion of cells possessing the specific disease marker and vice versa. So far, the correlation of results with the disease outcome confirmed that this simple semi-quantitative approach may in some cases substitute laborious precisely quantifying techniques in the monitoring of the disease. In optimized multiplex IgH/ras PCR co-amplifying clonal CDR3 rearrangement of IgH and the codon 61 of Hras 1 gene, the amount of CDR3 product as the disease marker is related to the ras product as a standard marker of all cells, and quantitative results are obtained by software analyses of detecting gels. Presumably, both approaches may provide clinically useful information on the disease activity and treatment outcome.

[Semiquantitative monitoring of molecular markers in non-Hodgkin's lymphoma]. / Semikvantitativní sledování molekulárních markeru u nehodgkinských lymfomu.

BACKGROUND: As quantitative changes of disease specific molecular markers may reflect disease activity, various methods quantifying targets of polymerase chain reaction were developed and their clinical relevance should be established. METHODS AND RESULTS: Here the exploitation of the DNA limiting dilution methodology in molecular monitoring of non-Hodgkin's lymphomas is presented. Long-term stored diagnostic DNAs are checked for their integrity and examined in dose-response assays for semiquantitative estimates of t(14,18) translocations and clonal immunoglobulin heavy-chain gene rearrangement. Assuming that the specific targets are diluted proportionally by dilution of total genomic DNA, sensitivities of polymerase chain reaction expressed as minimal amounts of total cells in reaction initiating positivity are compared. The term PCR-detectability as the ratio of sensitivities determined for preceding and actual samples is introduced. The value of PCR-detectability lower than one is considered as an indicator of a decrease of cells bearing the marker and vice versa. So far, the considerable increases in PCR-detectabilities were found close to relapses and unsubstantial changes at clinical remissions. CONCLUSIONS: It is presumable, that the semiquantitative limiting dilution approach may contribute to the monitoring of disease and treatment outcome.

Monoclonal antibodies to Escherichia coli beta-galactosidase and their use for detection and purification of recombinant expression products.

A panel of seven mouse monoclonal antibodies (BG-01-BG-07) was prepared against beta-galactosidase derived from E. coli. The antibodies are beta-galactosidase specific, show no cross-reactivity with other E. coli proteins and can be used for identification and characterization of beta-galactosidase fusion proteins expressed in lambda expression vectors. One of the antibodies allows a simple, one-step isolation of the fusion proteins directly from the crude bacterial lysates using immunoaffinity chromatography.

A novel panel of monoclonal antibodies against beta-galactosidase of Escherichia coli and its versatility for detection of recombinant expression products.

A panel of seven mouse monoclonal antibodies (BG-O1 - BG-07) raised against beta-galactosidase (beta-gal) from E. coli was characterized in respect of their binding to beta-gal and to fusion proteins. The antibodies were beta-gal specific, recognized six different antigenic determinants on beta-gal molecule and some of them inhibited catalytical activity of the enzyme. The antibodies reacted with C-terminal beta-gal fusion proteins in the native state as well as after Western blotting. BG-02 antibody was successfully used for immunofluorescence detection of cells transfected with vectors containing the lacZ gene.

Electrophoresis of nonreduced IgM and IgG monoclonal antibodies on mixed agarose-polyacrylamide gels.

A gel matrix composed of 0.5% agarose and 2.5% polyacrylamide allows the parallel electrophoretic separation of nonreduced IgM and IgG monoclonal antibodies. The mobilities of IgM and IgG differ at pH 9.5, which enables a preliminary Ig class designation. It allows rapid detection of incomplete cloning if both IgM and IgG are present. A semiquantitative evaluation of nonreduced IgM in ascitic fluid is possible as well.

[Parenteral nutrition using branched-chain amino acids (Nutramin VLI Spofa) in the early postoperative period in patients with intra-abdominal inflammatory diseases]. / Parenterální výziva s pouzitím rozvetvených aminokyselin (Nutramin VLI Spofa) v casném pooperacním období u nemocných s nitrobrisními zánetlivými onemocneními.

In 19 patients with intraabdominal septic conditions hospitalized at the Surgical Clinic of the Medical Faculty of Hygiene, Charles University, Prague 10 in 1987-1988, the effect of the gradually increasing ratio of branched amino acids administered in parenteral nutrition was investigated. The administration of branched amino acids (Nutramin VLI Spofa) was started already on the first day after operation. By increasing the ratio to 43% of branched amino acids gradual normalization of metabolic changes occurred, incl. normalization of free amino acid plasma levels.

Electrophoretic location of epitopes on human transferrin.

Competitive assays of antigen--antibody interaction using solid-bound antigen or antibodies can be affected by steric restrictions. A suitable electrophoretic system may be used to follow the interactions in solution. Electrophoresis of human transferrin complexed with pairs of monoclonal antibodies raised against it revealed three types of interaction: competition for closely neighbouring epitopes, simultaneous binding to distinct epitopes, and a type of interaction suggesting formation of multiply linked antigen--antibody complexes.