Quantitation of Minimal Residual Disease in Patients with Chronic Lymphocytic Leukemia Using Locked Nucleic Acid-Modified, Fluorescently Labeled Hybridization Probes and Real-Time PCR Technology

2007 ◽  
Vol 11 (5) ◽  
pp. 325-335 ◽  
Author(s):  
Soňa Peková ◽  
Ludmila Bezdîčková ◽  
Lukáš Smolej ◽  
Tomáš Kozák ◽  
Ivana Hochová ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Real Time Pcr ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Locked Nucleic Acid ◽  
Hybridization Probes ◽  
Pcr Technology ◽  
Minimal Residual

Quantitation of Minimal Residual Disease in Patients with Chronic Lymphocytic Leukemia Using LNA-Modified Fluorescently Labeled Probes and Real-Time PCR Technology.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2331-2331
Author(s):  
Sona Pekova ◽  
Ludmila Saudkova ◽  
Lukas Smolej ◽  
Miroslav Prucha ◽  
Tomas Kozak
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tumor Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Specific Primers ◽  
Pcr Technology ◽  
Minimal Residual

Abstract Background: Patients with chronic lymphocytic leukemia (CLL) relapse even after aggressive therapies and stem cell transplantation. As the therapeutical goal today is to clear off the tumor cell burden as much as possible, highly sensitive assays for minimal residual disease (MRD) evaluation and monitoring are needed. At present, many patients with not only germline IgVH sequences, but also with hypermutated IgVH genes are being treated, with the need for a sensitive and specific MRD monitoring. The original notion of MRD follow-up in CLL was based on the usage of JH-gene specific TaqMan hybridization probes. At present, due to the vast diversity of B-clonal rearrangements to be detected, the original idea has been challenged and the methodology should be modified. Aims: Since the hypermutation process does not restrict itself to the VH segments only and might afflict the JH segment as well, the molecular tools for the monitoring of CLL clonal rearrangements must be versatile enough to allow for the detection and quantitation of virtually any sequence possible. Moreover, the technique must meet the criteria for high sensitivity and specificity. We present here a novel methodology for MRD monitoring in CLL, based on LNA technology (Locked Nucleic Acids) and quantitative Real-Time PCR. Methods: 59 patients with the diagnosis of CLL were enrolled into our MRD study (22 females, 37 males, median age 59.1 yrs). 33 out of 59 individuals had unmutated IgVH genes (4 females, 29 males), 26 out of 59 patients had mutated IgVH genes (15 females, 11 males). For each patient, clone-specific primers were designed and their clonal VH sequences were molecularly cloned to construct the quantitation standards. In one patient, allelic inclusion has been identified (VH1–8 and VH3–30, both mutated), and for this individual, clone-specific primers and standards have been constructed for both rearrangements. To quantify the individual clonal VH transcripts, LNA-modified fluorescently labeled probes targeted against individual gene segments were employed. For any of 6 (7) IgVH families with unmutated VH genes, family-specific consensus LNA-modified probes were used. For those CLL cases with heavily hypermutated genes, ProbeLibrary™ was employed. For quantitation experiments, ABL was used as the control gene. Results: The LNA-modified probes are distinguished by a very high specificity and sensitivity (reaching to 10−8, in contrast to flow cytometry with its detection limit being 10−4). The LNA-based assays allow for precise monitoring of the residual tumor cell burden in CLL patients, especially during those periods of time, when other, less sensitive techniques fail to trace the malignant clone. Conclusions: LNA-modified probes and Real-Time PCR technology represent a highly versatile, specific and extremely sensitive methodology for the monitoring of MRD in chronic lymphocytic leukemia. We strongly advocate their usage in the molecular follow-up of MRD in the setting of CLL (and possibly other B-cell malignancies with hypermutated VH gene sequences as well).

scholarly journals Combination chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab shows significant clinical activity with low accompanying toxicity in previously untreated B chronic lymphocytic leukemia

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 405-411 ◽  
Author(s):  
Neil E. Kay ◽  
Susan M. Geyer ◽  
Timothy G. Call ◽  
Tait D. Shanafelt ◽  
Clive S. Zent ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Hematologic Toxicity ◽  
Color Flow ◽  
Significant Clinical Activity ◽  
Minimal Residual

Abstract Building on the prior work of use of pentostatin in chronic lymphocytic leukemia (CLL), we initiated a trial of combined pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) for 65 symptomatic, previously untreated patients. Of 64 evaluable patients, 34 (53%) were high Rai risk, 71% were nonmutated for the immunoglobulin heavy-chain variable region gene, 34% were CD38+, and 34% were ZAP-70+. Thirty patients (52%) had one anomaly detected by fluorescence in situ (FISH) hybridization, and 21 (36%) had complex FISH defects. Thirty-eight patients (58%) had grade 3+ hematologic toxicity but minimal transfusion needs and no major infections. Responses occurred in 58 patients (91%), with 26 (41%) complete responses (CRs), 14 (22%) nodular partial responses (nodular PRs), and 18 (28%) partial responses (PRs). Many patients with a CR also lacked evidence of minimal residual disease by 2-color flow cytometry. Examination of prognostic factors demonstrated poor response in the 3 patients with del(17p). In contrast, we found this regimen was equally effective in young versus older (> 70 years) patients and in del(11q22.3) versus other favorable prognostic factors. Thus, this novel regimen of pentostatin, cyclophosphamide, and rituximab for previously untreated patients with CLL demonstrated significant clinical activity despite poor risk-based prognoses, achievement of minimal residual disease in some, and modest toxicity.

Detection and impact of minimal residual disease on outcome of chronic lymphocytic leukemia

2012 ◽  
Vol 153 (41) ◽  
pp. 1622-1628
Author(s):  
Márk Plander ◽  
Judit Skrapits ◽  
Tünde Bozsó ◽  
Tamás Szendrei ◽  
János László Iványi
Keyword(s):  
Overall Survival ◽  
Chronic Lymphocytic Leukemia ◽  
Complete Remission ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Single Agent ◽  
Progression Free Survival ◽  
Lymphocytic Leukemia ◽  
Free Survival ◽  
Minimal Residual

Introduction: Minimal residual disease is associated with longer overall survival in patients with chronic lymphocytic leukemia. Aim: The aim of the authors was to determine the clinical significance of remission and minimal residual disease on the survival of patients with chronic lymphocytic leukemia. Methods: Data from 42 first-line treated patients with chronic lymphocytic leukemia were analyzed. Minimal residual disease was determined by flow cytometry. Results: Overall response and complete remission was achieved in 91%, 86%, 100% and 87%, 0%, 60% of patients with fludarabine-based combinations, single-agent fludarabine and cyclophosphamide + vincristin + prednisolone regimen, respectively. Minimal residual disease eradication was feasible only with fludarabine-based combinations in 60% of these cases. The ratio of minimal residual disease was 0.5% on average. During a median follow-up period lasting 30 months, the overall survival of patients with fludarabine-resistant disease proved to be significantly shorter (p = 0.04), while complete remission without minimal residual disease was associated with significantly longer progression free survival (p = 0.02). Conclusion: Only fludarabine-based combinations were able to eradicate minimal residual disease in patients with chronic lymphocytic leukemia. Complete remission without minimal residual disease may predict longer progression free survival in these patients. Orv. Hetil., 2012, 153, 1622–1628.

scholarly journals Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program

2015 ◽  
Vol 139 (10) ◽  
pp. 1276-1280 ◽  
Author(s):  
Michael Keeney ◽  
Jaimie G. Halley ◽  
Daniel D. Rhoads ◽  
M. Qasim Ansari ◽  
Steven J. Kussick ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Plasma Cell Myeloma ◽  
Minimal Residual

Context Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. Objectives To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. Design Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry—Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. Results A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. Conclusions There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

Combination Chemotherapy with Pentostatin, Cyclophosphamide and Rituximab Induces High Rate of Remissions Including Complete Responses and Achievement of Minimal Residual Disease in Previously Untreated B-Chronic Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 339-339 ◽  
Author(s):  
Neil E. Kay ◽  
Susan M. Geyer ◽  
Thomas Lin ◽  
Timothy G. Call ◽  
Diane F. Jelinek ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Post Treatment ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Significant Activity ◽  
Color Flow ◽  
Minimal Residual

Abstract The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., >90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.

Treatment of Younger Patients with Chronic Lymphocytic Leukemia

Hematology ◽  
2010 ◽  
Vol 2010 (1) ◽  
pp. 82-89 ◽  
Author(s):  
Alessandra Ferrajoli
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Consolidation Therapy ◽  
Newly Diagnosed ◽  
Therapeutic Approaches ◽  
Early Referral ◽  
Younger Patients ◽  
Minimal Residual

Abstract Younger patients (defined as patients younger than 50–55 years of age) represent a small group of newly diagnosed patients with chronic lymphocytic leukemia, accounting only for 10% to 20% of newly diagnosed cases. However, once these patients become symptomatic and require treatment, their life expectancy is significantly reduced. Therapeutic approaches for younger patients should be directed at improving survival by achieving a complete remission and, where possible, eradicating minimal residual disease. Chemoimmunotherapy combinations carry the highest response rates and are commonly offered to younger patients. Additional strategies that should be considered for younger patients include early referral for stem-cell transplantation and clinical trials of consolidation therapy to eliminate minimal residual disease.

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