Different Subclones of Adult Acute Lymphoblastic Leukemia Detected by Clonal Igh and TCR Markers Behave Differently during Remission and at Relapse

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4860-4860
Author(s):  
Yulya Davidyan ◽  
Elena Parovichnikova ◽  
Vadim Surin ◽  
Valeryi Savchenko
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Gene Rearrangement ◽  
Lymphoblastic Leukemia ◽  
False Negative ◽  
Patient Specific ◽  
Gene Rearrangements ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Abstract INTRODUCTION. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient–specific targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). However they might be unstable during the disease course. False-negative results due to clonal evolution are the major disadvantage of using Ig/TCR gene rearrangements as PCR targets for MRD detection. In order to minimize false-negative results, it is necessary to use more PCR targets for MRD monitoring. PATIENTS AND METHODS. Bone marrow samples of 34 ALL patients (27 B-ALL, 7 T-ALL) were analyzed for IgH and TCR gene rearrangement by means of PCR method at diagnosis, after induction and consolidation of remission, before and after stem cell transplantation and during maintenance therapy. RESULTS. Clonal IgH and TCR gene targets for MRD detection were identified in 97% of the patients (33/34). In 70% of the patients (23/33) two or more specific markers for MRD monitoring were detected. Three clonal IgH gene rearrangements were discovered in 2 patients. We wished to investigate whether it was important to monitor two or more targets, because of the dynamics of various markers (subclones) may be different). Prolonged MRD monitoring with 2–3 markers was carried out in 9 patients on different stages of treatment. In 4 of 9 patients (3 precursor B-ALL, 1 pre-T-ALL) the results of MRD detection by 2–3 clonal markers at different time points were similar. And in 5 of 9 patients (3 precursor-B-ALL, 1 precursor T-ALL, 1 B-mature ALL) marker detectability during treatment was dissimilar and not parallel. Of note, 4 of those 5 patients with varying dynamics of different subclones relapsed. Relapse was not observed in the B-mature ALL patient. No relapses were detected in 4 patients with similar dynamics of MRD markers. Bone marrow samples of 8 adult ALL patients were subjected to a detailed analysis of IgH and TCR gene rearrangements by PCR method at diagnosis and relapse. In most patients (7/8) rearrangements identified at diagnosis were present also at relapse. In one patient with precursor-B-ALL one clonal incomplete IgH gene rearrangement (DH-JH) detected at diagnosis was lost at relapse and another one DH-JH rearrangement was preserved. Of note, acute myeloid leukemia was diagnosed at relapse in this patient. CONCLUSIONS. Comparative PCR analyses of IgH and TCR gene rearrangements did not detect differences between newly diagnosed and relapsed ALL although in a small group of adult patients (unlike childhood ALL). Our data indicate the necessity to monitor MRD in ALL patients with more than one PCR target in order to minimize false-negative results and to predict the course of the disease. The detectability of various MRD markers (subclones) during treatment may differ and this fact could predict a higher probability of relapse.

The Genomic Landscape of PAX5, IKZF1, and CDKN2A/B Alterations in B-Cell Precursor Acute Lymphoblastic Leukemia

2016 ◽  
Vol 150 (3-4) ◽  
pp. 242-252 ◽  
Author(s):  
Zhishuo Ou ◽  
Maureen Sherer ◽  
Jane Casey ◽  
Heather A. Bakos ◽  
Kathleen Vitullo ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
False Negative ◽  
Prognostic Significance ◽  
Cell Precursor ◽  
Genomic Alterations ◽  
Acgh Analysis ◽  
Negative Results ◽  
False Negative Results

We present a comprehensive comparison of PAX5,IKZF1, and CDKN2A/B abnormalities in 21 B-cell precursor acute lymphoblastic leukemia (B-ALL) patients studied by aCGH and gene-specific FISH assays. In our cohort of B-ALL patients, alterations of IKZF1, PAX5, and CDKN2A/B were detected by aCGH analysis in 43, 52, and 57% of samples, respectively. Deletions of IKZF1 were present in 9 samples, including 5 cases positive for both PAX5 and IKZF1 deletions, implying digenic impairment. Furthermore, all cases with IKZF1 deletions also had additional genomic alterations, including BCR-ABL1 gene fusions, PAX5 deletions, CDKN2A/B deletions, and FLT3 amplification. Deletions of CDKN2A/B represented the most frequent abnormalities in our group of patients. Our study demonstrates the high incidence of PAX5, IKZF1, and CDKN2A/B alterations in B-ALL detected by aCGH analysis. Due to the small size and variability in the deletion breakpoints, FISH studies showed false-negative results in 10, 40, and 28% of the samples tested for the IKZF1,PAX5, and CDKN2A/B gene deletions, respectively. The PAX5 and IKZF1 abnormalities are highly specific to B-ALL and can be used as diagnostic markers. Moreover, IKZF1 alterations frequently coexist with a BCR-ABL gene fusion. Our study revealed multiple additional B-ALL-specific genomic alterations and showed that aCGH is a more sensitive method than FISH, allowing whole genome profiling and identification of aberrations of diagnostic and prognostic significance in patients with B-ALL.

scholarly journals Validation of DNA Methylation Biomarkers for Diagnosis of Acute Lymphoblastic Leukemia

2014 ◽  
Vol 60 (7) ◽  
pp. 995-1003 ◽  
Author(s):  
Zac Chatterton ◽  
Daniel Burke ◽  
Kerry R Emslie ◽  
Jeffery M Craig ◽  
Jane Ng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Limit Of Detection ◽  
Lymphoblastic Leukemia ◽  
False Negative ◽  
Disease Diagnosis ◽  
Patient Specific ◽  
Bone Marrow Biopsies ◽  
Maldi Tof

Abstract BACKGROUND DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing. METHODS We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods. RESULTS Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000–5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients. CONCLUSIONS Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

scholarly journals Assessment of individual molecular response in chronic myeloid leukemia patients with atypical BCR-ABL1 fusion transcripts: recommendations by the EUTOS cooperative network

2021 ◽  
Author(s):  
Vivien Schäfer ◽  
Helen E. White ◽  
Gareth Gerrard ◽  
Susanne Möbius ◽  
Susanne Saussele ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
False Negative ◽  
Patient Specific ◽  
Molecular Response ◽  
Real Time Quantitative Pcr ◽  
Log Reduction ◽  
Negative Results ◽  
False Negative Results

Abstract Purpose Approximately 1–2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients. Methods BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR). Results In total, 330 blood samples (2–34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios. Conclusions Characterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.

scholarly journals Rearrangement of the T cell receptor gamma-chain gene in childhood acute lymphoblastic leukemia

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1933-1939
Author(s):  
A Tawa ◽  
SH Benedict ◽  
J Hara ◽  
N Hozumi ◽  
EW Gelfand
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
T Cell Receptor ◽  
Gene Rearrangement ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Cell Receptor ◽  
Gene Rearrangements ◽  
Gamma Chain ◽  
Beta Gene

We analyzed rearrangements of the T cell receptor gamma-chain (T gamma) gene as well as rearrangements of the T cell receptor beta-chain (T beta) gene and immunoglobulin heavy-chain (IgH) gene in 68 children with acute lymphoblastic leukemia (ALL). All 15 patients with T cell ALL showed rearrangements of both T beta and T gamma genes. Twenty-four of 53 non-T, non-B ALL patients (45%) showed T gamma gene rearrangements and only 14 of these also showed T beta gene rearrangements. Only a single patient rearranged the T beta gene in the absence of T gamma gene rearrangement. The rearrangement patterns of the T gamma gene in non-T, non-B ALL were quite different from those observed in T cell ALL, as 20 of 23 patients retained at least one germline band of the T gamma gene. In contrast, all T cell ALL patients showed no retention of germline bands. These data indicate that rearrangement of the T gamma gene is not specific for T cell ALL. Further, the results also suggest that T gamma gene rearrangement precedes T beta gene rearrangement. The combined analysis of rearrangement patterns of IgH, T beta, and T gamma genes provides new criteria for defining the cellular origin of leukemic cells and for further delineation of leukemia cell heterogeneity.

Development of a recombinase polymerase amplification assay forVibrio parahaemolyticusdetection with an internal amplification control

2018 ◽  
Vol 64 (4) ◽  
pp. 223-230 ◽  
Author(s):  
Huan-Lan Yang ◽  
Shuang Wei ◽  
Ravi Gooneratne ◽  
Anthony N. Mutukumira ◽  
Xue-Jun Ma ◽  
...  
Keyword(s):  
Bacterial Species ◽  
False Negative ◽  
Recombinase Polymerase Amplification ◽  
Internal Amplification Control ◽  
Target Sequence ◽  
Negative Results ◽  
False Negative Results ◽  
Amplification Assay ◽  
Pcr Method ◽  
Recombinase Polymerase Amplification Assay

A novel RPA–IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA–IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

Minimal residual disease in acute lymphoblastic leukemia detected by immune selection and gene rearrangement analysis.

1989 ◽  
Vol 7 (3) ◽  
pp. 338-343 ◽  
Author(s):  
M Bregni ◽  
S Siena ◽  
A Neri ◽  
R Bassan ◽  
T Barbui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Minimal Residual Disease ◽  
Gene Rearrangement ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Immune Selection ◽  
Gene Rearrangement Analysis ◽  
Minimal Residual

We have developed an assay for the detection of malignant residual cells in the bone marrow from patients with B- or T-lineage acute lymphoblastic leukemia (ALL) in clinical remission. This assay involves an immune selection step followed by immunoglobulin or T-cell receptor gene rearrangement analysis and allows the detection of one contaminating tumor cell out of 1,000 normal bone marrow cells. We have examined the bone marrow of 11 patients with adult ALL in remission over a 24-month period. Five patients relapsed in the bone marrow and one in the CNS. The assay allowed the detection of minimal residual disease in four of five patients that subsequently relapsed in the bone marrow, 1.5 to 9 months before the relapse became morphologically and clinically manifest. Residual disease was not found in the bone marrow from patients in continuous remission and from the single patient who relapsed in the CNS. We conclude that the ability of the assay described here to detect minimal residual disease with high specificity can provide information for further understanding of the biology of ALL and hopefully for the clinical management of patients with this disease.

scholarly journals Evolution of Tumor Clones in Adult Acute Lymphoblastic Leukemia

Acta Naturae ◽  
2016 ◽  
Vol 8 (4) ◽  
pp. 100-109 ◽  
Author(s):  
S. Yu. Smirnova ◽  
Yu. V. Sidorova ◽  
N. V. Ryzhikova ◽  
K. A. Sychevskaya ◽  
E. N. Parovichnikova ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Disease Onset ◽  
Disease Recurrence ◽  
Detection Sensitivity ◽  
Patient Specific ◽  
Gene Rearrangements ◽  
Childhood All

Clonal instability of a tumor cell population in acute lymphoblastic leukemia (ALL) may complicate the monitoring of a minimal residual disease (MRD) by means of patient-specific targets identified at the disease onset. Most of the data concerning the possible instability of rearranged clonal TCR and IG genes during disease recurrence were obtained for ALL in children. The appropriate features of adult ALL, which are known to differ from those of childhood ALL in certain biological characteristics and prognosis, remain insufficiently studied. The aim of this study was to assess the stability of IG and TCR gene rearrangements in adult ALL. Rearrangements were identified according to the BIOMED-2 protocol (PCR followed by fragment analysis). Mismatch in clonal rearrangements at onset and relapse was identified in 83% of patients, indicating clonal instability during treatment. Clonal evolution and diversity of IG and TCR gene rearrangements may be one of the tumor progression mechanisms. New rearrangements may emerge due to residual VDJ-recombinase activity in tumor cells. Also, many clonal IG and TCR gene rearrangements may be present at different levels at a diagnosis, but less abundant clones may be invisible due to limited detection sensitivity. Later, major clones may disappear in the course of chemotherapy, while others may proliferate. Investigation of clonal evolution and heterogeneity in ALL and their impact on the treatment efficacy will contribute to the identification of new prognostic factors and the development of therapeutic approaches.

Nonhematological Tumor Metastasis in the Bone Marrow: An Analysis of 10112 Unselected Plastic-Embedded Biopsy Sections.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5154-5154
Author(s):  
Xiao Li ◽  
Ying Tao ◽  
Yizhi Liu ◽  
Luxi Song ◽  
Lingyun Wu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
False Negative ◽  
Metastatic Carcinoma ◽  
Special Focus ◽  
Metastatic Tumors ◽  
Primary Tumors ◽  
Negative Results ◽  
False Negative Results ◽  
Tumor Metastases ◽  
Biopsy Examination

Abstract We investigated the frequency of bone marrow (BM) involvement by nonhematological tumors in unselected BM biopsy samples from 10,112 patients. Plastic embedded BM biopsy sections were retrospectively analyzed, with special focus on Frequency of BM involvement by nonhematological malignancies, the advantage of plastic-embedded BM biopsy specimens over paraffin-embedded samples, Complementarity of marrow biopsy to aspiration smears. the causes of false-negative results when using aspiration smears, and analysis of primary sites of metastatic solid tumors. Overall 101 of the 10112 BM samples showed nonhematological tumor metastases, accounting for 1.0% of all analyzed specimens, which was higher than data reported on the basis of paraffin embedded samples. Of 74 aspiration smears obtained concurrently with the 101 biopsy sections, 55 were also positive for metastatic carcinoma. That is, the detection rate of metastatic tumors based on the aspiration smears was 74.3% relative to the rate observed on biopsy sections. All 101 sections with metastatic tumors showed various degrees of myelofibrosis (including 67cases with severe myelofibrosis [+++]) which was thought to be partly responsible for false negative results on aspiration samples. Primary tumors were documented ante-mortem in 50 of the 101 BM biopsy-positive patients (49.5%). The most frequent primary sites were lung, gastroinyestinal tract, and breast, in good agreement with data in the literature. In conclusion, nonhematological tumor metastases in unselected bone marrow are not exceptional findings. Plastic embedded BM biopsy examination is helpful in detecting unknown metastatic nonhematological malignancies and apparently more sensitive than paraffin embedded BM biopsies. Figure Figure Figure Figure

Dynamics and Evolution of the Preleukemic Clone in Slow Onset Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1306-1306
Author(s):  
Olga Zimmermannova ◽  
Ondrej Hrusak ◽  
Eva Fronkova ◽  
Marketa Kubricanova Zaliova ◽  
Ester Mejstrikova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Gene Rearrangement ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Snp Array ◽  
Gene Rearrangements ◽  
T Cell Clone ◽  
Leukemic Clone ◽  
Diagnostic Samples

Abstract Abstract 1306 Introduction Acute lymphoblastic leukemia (ALL) can rarely be manifested before its diagnosis as pancytopenia and bone-marrow aplasia followed by spontaneous transient hematological remission. A leukemic clone can be detected during this particular period, making it possible to follow its dynamics in size, as well as independent changes leading to the overt disease. Patients and methods We initially selected 5 cases of ALL, in which the pre-diagnostic period of anemia had occurred. In all pre-diagnostic samples, the immunoglobulin (Ig) and/or T-cell receptor (TCR) gene rearrangements were investigated using quantitative real-time PCR. In addition, SNP array and FISH were performed in some cases in order to reveal other aberrations specific to leukemia. To backtrack the preleukemic cells three different approaches were employed - qPCR, FISH and next generation sequencing (NGS). Results Immunoreceptor gene rearrangements were detected in all 5 patients in the diagnostic sample. At least one of the diagnostic rearrangements was found in all preleukemic bone-marrow samples. The levels of positivity differed from 10−1 to 10−6. In 3 patients more than one pre-diagnostic sample was made available for our examination. A surprising dynamics in the size of the leukemic clone was revealed by the investigation. Although a steady increase of blasts was expected, particularly high levels occurred in the first available preleukemic samples (10% and more clonal Ig/TCR positive cells compared to the diagnosis, in 4/5 of patients), followed by remarkable decrease 1 to 3 months before the diagnosis in 3 patients with more than one sample available. This dynamics points to possible immune system interventions during preleukemic phase. In 2 patients NGS (targeted to Ig/TCR gene rearrangements, Adaptive Biotechnologies, Seattle) was applied. The acquired data correlated with the results of qPCR backtracking. In addition, more detailed information was gained about gene rearrangement spectra as well as about possible immunological background of the leukemia progression. In both these patients, the examination revealed a significant fraction of non-malignant γΔ T cell-clone carrying an identical Vg9-Jg1.2 gene rearrangement, which has been reported previously to play an important role in anti-tumour and anti-microbe immune responses. Interestingly, the dynamics of this particular clone seemed to correlate negatively with the leukemic cell levels, pointing out its putative role in tumour surveillance. Consequently, SNP array was performed to define the aberrations acquired, followed by FISH investigation to confirm the above conclusions. While some aberrations were confirmed already in the pre-diagnostic samples (namely ETV6 deletion in the patient with ETV6/RUNX1 positive ALL), others occurred in the diagnostic sample, yet not in the preleukemic cells several months before the diagnosis. Conclusion Preleukemic cells present during the aplastic phase preceding leukemia onset are not just a gradually growing clone. Their specific dynamics has been shown, which supports our previous data acquired during the investigation of secondary leukemias. Furthermore, at least in some cases, this clone indeed should be considered ‘preleukemic’ as it is to undergo at least one more hit or subclonal selection to progress into an active disease. Disclosures: No relevant conflicts of interest to declare.

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