Evaluation of Minimal Residual Disease Using Reverse-Transcription Polymerase Chain Reaction in t(8;21) Acute Myeloid Leukemia: A Multicenter Study of 51 Patients

2000 ◽  
Vol 18 (4) ◽  
pp. 788-788 ◽  
Author(s):  
F. Morschhauser ◽  
J.M. Cayuela ◽  
S. Martini ◽  
A. Baruchel ◽  
P. Rousselot ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Residual Disease ◽  
Rank Test ◽  
Rt Pcr ◽  
Log Rank Test ◽  
One Step ◽  
Polymerase Chain ◽  
The One ◽  
Pcr Techniques

PURPOSE: Most studies using various reverse-transcription polymerase chain reaction (RT-PCR) techniques reported that the detection of the AML1-ETO fusion transcript was a common finding in long-term complete remission (CR) in acute myeloid leukemia (AML) with t(8;21) translocation. However, larger prospective studies with interlaboratory quality control may be important to investigate more precisely the clinical usefulness of studying minimal residual disease with RT-PCR in t(8;21) AML. PATIENTS AND METHODS: We collected 223 marrow samples from 51 patients with t(8;21) AML diagnosed in five centers and tested all samples by two different RT-PCR techniques (a nested technique and a one-step technique, with a sensitivity of 10−6 and 10−5, respectively) in two different laboratories. RESULTS: Samples from 14 patients in long persistent CR (median follow-up duration, 112 months) were taken at least twice, and all were PCR-negative by both techniques. Samples were prospectively taken from 37 patients after achievement of first CR and/or second CR, before intensive consolidation treatment, and every 3 to 6 months after completion of therapy. Patients who converted to PCR negativity with the one-step technique (60%) or both techniques (48%) after CR achievement had a longer CR duration than those with persistently positive PCR results (two-sided log-rank test, P = .0001). Patients who became PCR-negative with the one-step technique before intensive consolidation (23%) had a lower relapse rate (11% v 72%) and a longer CR duration than those who remained persistently PCR-positive at that point (two-sided log-rank test, P = .0015). CONCLUSION: Patients with AML with t(8;21) in long-term remission were all PCR-negative. In prospectively studied patients, a good correlation was found between negative PCR results and absence of relapse. Early negative results with the one-step RT-PCR technique, before consolidation treatment, seemed to carry an especially good prognosis, suggesting that RT-PCR analysis could help in choosing the type of consolidation therapy in patients with t(8;21) AML.

scholarly journals Absence of reverse transcription-polymerase chain reaction detectable residual disease in patients with acute promyelocytic leukemia in long- term remission

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3556-3559 ◽  
Author(s):  
D Diverio ◽  
PP Pandolfi ◽  
A Biondi ◽  
G Avvisati ◽  
MC Petti ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Clinical Relevance ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Hybrid fusion genes are specific tumor markers of several leukemic subtypes. The use of reverse transcription-polymerase chain reaction (RT-PCR) to amplify chimeric cDNAs allows sensitive detection of the neoplastic clone for diagnostic and monitoring studies in these leukemias. Nonetheless, the clinical relevance of minimal residual disease (MRD) evaluation by PCR remains controversial. In this study, 9 patients (pts) with acute promyelocytic leukemia (APL) in long-term remission for 4 to 12 years were analyzed for the presence of MRD by RT- PCR amplification of the specific PML/RAR-alpha fusion gene. Seven pts had been treated with conventional chemotherapy (CHT) alone, 1 had undergone allogeneic bone marrow transplantation (BMT), and 1 autologous BMT as consolidation therapy after CHT. In 8 cases, the presence of the t(15;17) rearrangement could be documented in diagnostic BM specimens by cytogenetic and/or molecular analysis. A two- rounds “nested” RT-PCR assay with sensitivity levels of 1 in 10(5) was used to analyze BM samples collected at 32 to 141 months from the achievement of complete remission (CR). In no cases were residual PML/RAR-alpha transcripts detectable in these remission controls. All patients are in unmaintained CR at 48 to 154 months from CR and at 6 to 17 months from PCR evaluation. These results suggest that long-term survival of APL is associated with eradication of cells carrying the specific PML/RAR-alpha rearrangement, indicating that PCR negativity should be considered the therapeutic goal in these patients. Our findings further strengthen the clinical relevance of PCR monitoring studies in APL, as opposite to other leukemic subtypes (chronic myeloid leukemia and acute myeloid leukemia-M2) in which the prognostic significance of PCR evaluation is unclear.

scholarly journals Absence of reverse transcription-polymerase chain reaction detectable residual disease in patients with acute promyelocytic leukemia in long- term remission

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3556-3559 ◽  
Author(s):  
D Diverio ◽  
PP Pandolfi ◽  
A Biondi ◽  
G Avvisati ◽  
MC Petti ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Clinical Relevance ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Hybrid fusion genes are specific tumor markers of several leukemic subtypes. The use of reverse transcription-polymerase chain reaction (RT-PCR) to amplify chimeric cDNAs allows sensitive detection of the neoplastic clone for diagnostic and monitoring studies in these leukemias. Nonetheless, the clinical relevance of minimal residual disease (MRD) evaluation by PCR remains controversial. In this study, 9 patients (pts) with acute promyelocytic leukemia (APL) in long-term remission for 4 to 12 years were analyzed for the presence of MRD by RT- PCR amplification of the specific PML/RAR-alpha fusion gene. Seven pts had been treated with conventional chemotherapy (CHT) alone, 1 had undergone allogeneic bone marrow transplantation (BMT), and 1 autologous BMT as consolidation therapy after CHT. In 8 cases, the presence of the t(15;17) rearrangement could be documented in diagnostic BM specimens by cytogenetic and/or molecular analysis. A two- rounds “nested” RT-PCR assay with sensitivity levels of 1 in 10(5) was used to analyze BM samples collected at 32 to 141 months from the achievement of complete remission (CR). In no cases were residual PML/RAR-alpha transcripts detectable in these remission controls. All patients are in unmaintained CR at 48 to 154 months from CR and at 6 to 17 months from PCR evaluation. These results suggest that long-term survival of APL is associated with eradication of cells carrying the specific PML/RAR-alpha rearrangement, indicating that PCR negativity should be considered the therapeutic goal in these patients. Our findings further strengthen the clinical relevance of PCR monitoring studies in APL, as opposite to other leukemic subtypes (chronic myeloid leukemia and acute myeloid leukemia-M2) in which the prognostic significance of PCR evaluation is unclear.

scholarly journals Detection of Cherry leaf roll virus and Strawberry latent ring spot virus by one-step RT-PCR

2009 ◽  
Vol 45 (No. 4) ◽  
pp. 140-143 ◽  
Author(s):  
S. Kumari
Keyword(s):  
Leaf Roll ◽  
Rt Pcr ◽  
Spot Virus ◽  
Cherry Leaf Roll Virus ◽  
One Step ◽  
Polymerase Chain ◽  
The One ◽  
Ring Spot ◽  
Screen House ◽  
Xiphinema Diversicaudatum

A one-step reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed and used for the detection of <i>Cherry leaf roll virus</i> (CLRV) and <i>Strawberry latent ring spot virus</i> (SLRSV). The protocol was used to test infected screen house plants and also plants from orchards and vineyards where the vector (<i>Xiphinema diversicaudatum</i>) of SLRSV was detected from the soil. The one-step RT-PCR protocol is rapid and sensitive and has the potential to be used for the diagnosis of CLRV and SLRSV in routine diagnostic laboratories.

scholarly journals A new method of "in-cell reverse transcriptase-polymerase chain reaction" for the detection of BCR/ABL transcript in chronic myeloid leukemia patients

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3822-3827 ◽  
Author(s):  
N Testoni ◽  
G Martinelli ◽  
P Farabegoli ◽  
A Zaccaria ◽  
M Amabile ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Single Cells ◽  
Fluorescent Microscopy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Methods of detecting minimal residual disease (MRD) in chronic myeloid leukemia (CML) include chromosome analysis, Southern blotting, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) techniques. We report a novel method to detect intracellular messenger RNA (mRNA) by combining the techniques of reverse transcription (RT) and PCR performed directly inside the cells, without extraction of the nucleic acid. We applied this method, which we call “in-cell RT-PCR”, to detect hybrid BCR/ABL transcript within single cells. After cellular permeabilization and fixation of single cells in suspension, the neoplastic mRNA was reverse transcribed into cDNA, and the cDNA was amplified by PCR with fluorescent primers, specific for bcr/abl. Flow cytometry was used to detect cells positive for the amplified DNA within the cell cytoplasm. After transferring the amplified cells onto slides by cytospin, the positive cells for BCR/ABL cDNA were observed by fluorescent microscopy. The technique was capable of detecting low abundancy signals and distinguishing different levels of gene expression. The amplification products were found in the cells and supernatants. The distribution was critically affected by the protease digestion condition. The specificity of amplification was confirmed by a nested RT-PCR of BCR/ABL performed on extracted mRNA from the same sample, and by reamplification of supernatants. We have used the technique to study 10 Ph+ CML patients and three normal subjects as controls. Four patients were 100% Ph+ at diagnosis time and RT-PCR+ at cytogenetic and molecular analysis, respectively. In-cell RT- PCR showed that the residual non-neoplastic cells could be observed in all cases. In two patients undergoing interferon-alpha (IFN-alpha) therapy and in four bone-marrow transplanted patients, the in-cell RT- PCR was used to compare the level of Ph+ positivity detected by cytogenetic analysis with the number of cells expressing BCR/ABL transcript. In this manner, we could estimate the MRD. Our preliminary application of the technique suggests that it is capable of accurately identifying cells transcribing bcr/abl, and that it may have significant clinical applications in the detection of MRD.

Competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction for quantitative assessment of minimal residual disease during postremission therapy in acute myeloid leukemia with inversion(16): a pilot study.

1998 ◽  
Vol 16 (4) ◽  
pp. 1519-1525 ◽  
Author(s):  
K Laczika ◽  
M Novak ◽  
B Hilgarth ◽  
M Mitterbauer ◽  
G Mitterbauer ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Rt Pcr ◽  
Molecular Monitoring ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Postremission Therapy ◽  
Minimal Residual ◽  
Acute Myeloid

PURPOSE (1) Quantification of minimal residual disease (MRD) by competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction (RT-PCR) in patients with acute myeloid leukemia (AML) and inversion(16) [inv(16)] during postremission therapy, (2) comparison of this method with conventional two-step RT-PCR, and (3) evaluation of a potential prognostic value. PATIENTS AND METHODS MRD of six consecutive adult patients with AML and inv(16)(p13;q22) or t(16;16)(p13;q22) who entered complete remission (CR) was monitored by competitive CBFbeta/MYH11 RT-PCR in their bone marrow (BM) during postremission therapy with high-dose cytarabine (HiDAC) or after BM transplantation with a matched unrelated-donor marrow (MUD-BMT) during an observation period of 4.5 to 27 months after initiation of treatment. RESULTS Competitive PCR showed a gradual decline by at least 4 orders of magnitude after 7 to 9 months in patients in continuous CR (CCR), while one patient who relapsed after 13.5 months only achieved a reduction by 2 orders of magnitude at the end of consolidation therapy. A rapid decrease below the detection limit was observed within 1 month in two patients after MUD-BMT. A temporary reappearance of molecular MRD was observed in these patients during immunosuppression for graft-versus-host disease (GvHD). After reduction of immunosuppression, the level of MRD dropped again below the PCR detection limit. Molecular monitoring by conventional two-step RT-PCR yielded comparable results only when multiple assays per time point were performed, while single-assay RT-PCR gave misleading results. CONCLUSION Competitive RT-PCR is a valuable tool for molecular monitoring during postremission chemotherapy, as well as after BMT.

scholarly journals Clinical significance of bcr-abl gene rearrangement detected by polymerase chain reaction after allogeneic bone marrow transplantation in chronic myelogenous leukemia

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2759-2767 ◽  
Author(s):  
R Delage ◽  
RJ Soiffer ◽  
K Dear ◽  
J Ritz
Keyword(s):  
Tumor Cells ◽  
Residual Disease ◽  
Residual Tumor ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Rank Test ◽  
Chain Reaction ◽  
Allogeneic Bmt ◽  
Log Rank Test ◽  
Polymerase Chain

Abstract In chronic myelogenous leukemia (CML), amplification of a segment of bcr-abl messenger RNA (mRNA) by polymerase chain reaction (PCR) can be used to detect minimal residual disease after bone marrow transplantation (BMT). Previous studies have shown that this sensitive technique can often detect small numbers of leukemia cells in patients who are otherwise in complete remission. Nevertheless, the clinical significance of PCR positivity remains unclear because the majority of patients with PCR-detectable bcr-abl mRNA can remain disease-free for prolonged periods after allogeneic BMT. In the present studies, we applied PCR to detect bcr-abl-positive cells in 100 serial blood or BM samples from 24 patients with CML who underwent CD6 T-cell-depleted allogeneic BMT. After BMT, bcr-abl mRNA could be detected in 20 patients (83.3%) during complete cytogenetic or clinical remission. Patients in whom PCR positivity was sustained over time had a higher probability of CML relapse than patients in whom PCR was intermittently negative (P = .0095, log rank test). PCR detection of bcr-abl transcript between 2 and 10 weeks post-BMT also was associated with a high probability of subsequent relapse (P = .023, log rank test). In eight selected patients, we used a titration assay of the PCR-amplified product to estimate the number of residual tumor cells in each clinical sample post-BMT. PCR results in four patients showed a continuing increase in the number of tumor cells from early posttransplant until either cytogenetic or clinical relapse could be detected by conventional methods 1 to 2 years later. In contrast, PCR detected either no leukemia cells or relatively low and stable numbers of residual tumor cells throughout the follow-up period in four patients who remained in clinical remission. These results show that detection of the bcr-abl transcript by PCR after allogeneic BMT in patients with CML has important prognostic value. Estimation of the number of tumor cells in serial analyses can also be used to detect proliferation of the residual leukemic population. Sensitive detection of minimal residual disease can be used to assess the effectiveness of the transplant preparative regimen and to direct and evaluate further therapy post-BMT, before the development of overt relapse.

scholarly journals The effect of chemo- and chemoimmunotherapy on the presence of circulating melanoma cells in peripheral blood. Preliminary results.

1998 ◽  
Vol 45 (1) ◽  
pp. 95-102 ◽  
Author(s):  
J Szenajch ◽  
A Kozak ◽  
A A Kruszewski ◽  
E Babiej ◽  
M Chomicka ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Advanced Disease ◽  
Residual Disease ◽  
Melanoma Cells ◽  
Response To Therapy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
The One ◽  
Therapy Protocol

Reverse transcription and polymerase chain reaction (RT/PCR) with primers specific for tyrosinase allow for a new method of early detection of individual melanoma cells in peripheral blood. Using this test the effect of chemo- and chemoimmunotherapy on the spread of early micrometastatic cancer cells has been evaluated. No significant correlations have been found between RT/PCR results on the one hand and stage of disease, a kind of the therapy protocol used and usage of the therapy as an adjuvant or palliative on the other hand. Thus, although the RT/PCR test for detection of circulating individual melanoma cells might help in identification of minimal residual disease in some patients, it has no application for routine staging of more advanced disease and in monitoring the response to therapy.

scholarly journals Clinical significance of bcr-abl gene rearrangement detected by polymerase chain reaction after allogeneic bone marrow transplantation in chronic myelogenous leukemia

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2759-2767 ◽  
Author(s):  
R Delage ◽  
RJ Soiffer ◽  
K Dear ◽  
J Ritz
Keyword(s):  
Tumor Cells ◽  
Residual Disease ◽  
Residual Tumor ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Rank Test ◽  
Chain Reaction ◽  
Allogeneic Bmt ◽  
Log Rank Test ◽  
Polymerase Chain

In chronic myelogenous leukemia (CML), amplification of a segment of bcr-abl messenger RNA (mRNA) by polymerase chain reaction (PCR) can be used to detect minimal residual disease after bone marrow transplantation (BMT). Previous studies have shown that this sensitive technique can often detect small numbers of leukemia cells in patients who are otherwise in complete remission. Nevertheless, the clinical significance of PCR positivity remains unclear because the majority of patients with PCR-detectable bcr-abl mRNA can remain disease-free for prolonged periods after allogeneic BMT. In the present studies, we applied PCR to detect bcr-abl-positive cells in 100 serial blood or BM samples from 24 patients with CML who underwent CD6 T-cell-depleted allogeneic BMT. After BMT, bcr-abl mRNA could be detected in 20 patients (83.3%) during complete cytogenetic or clinical remission. Patients in whom PCR positivity was sustained over time had a higher probability of CML relapse than patients in whom PCR was intermittently negative (P = .0095, log rank test). PCR detection of bcr-abl transcript between 2 and 10 weeks post-BMT also was associated with a high probability of subsequent relapse (P = .023, log rank test). In eight selected patients, we used a titration assay of the PCR-amplified product to estimate the number of residual tumor cells in each clinical sample post-BMT. PCR results in four patients showed a continuing increase in the number of tumor cells from early posttransplant until either cytogenetic or clinical relapse could be detected by conventional methods 1 to 2 years later. In contrast, PCR detected either no leukemia cells or relatively low and stable numbers of residual tumor cells throughout the follow-up period in four patients who remained in clinical remission. These results show that detection of the bcr-abl transcript by PCR after allogeneic BMT in patients with CML has important prognostic value. Estimation of the number of tumor cells in serial analyses can also be used to detect proliferation of the residual leukemic population. Sensitive detection of minimal residual disease can be used to assess the effectiveness of the transplant preparative regimen and to direct and evaluate further therapy post-BMT, before the development of overt relapse.

scholarly journals Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Elena O. Vidyagina ◽  
Nikolay N. Kharchenko ◽  
Konstantin A. Shestibratov
Keyword(s):  
Survival Rate ◽  
Axillary Buds ◽  
Long Term Storage ◽  
Average Survival ◽  
Transgenic Lines ◽  
Ssr Loci ◽  
One Step ◽  
The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

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