Ingrid Thörn

Minimal residual disease assessment in childhood acute lymphoblastic leukaemia: a Swedish multi-centre study comparing real-time polymerase chain reaction and multicolour flow cytometry.

Minimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi‐centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow‐up samples in 228 children using real‐time quantitative polymerase chain reaction (RQ‐PCR) of rearranged immunoglobulin/T‐cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0·1%, which was the sensitivity level reached in all analyses, the concordance between RQ‐PCR and FCM MRD values at day 29 was 84%. In B‐cell precursor ALL, an MRD level of ≥0·1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ‐PCR, a higher MRD cut‐off (≥0·2%) improved the predictive capacity of RQ‐PCR. In T‐ALL, RQ‐PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ≥0·1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ‐PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.

Both CD4(+) FoxP3(+) and CD4(+) FoxP3(-) T cells from patients with B-cell malignancy express cytolytic markers and kill autologous leukaemic B cells in vitro.

Cytotoxic CD4+ T cells have been found in patients with chronic lymphocytic leukaemia (CLL) and seem to be involved in the regulation of malignant B cells. The CD4+ T regulatory cells (Tregs) can regulate various immune cells, including B cells, by inducing their apoptosis. Hence, different subgroups of CD4+ T cells may be involved in the regulation of malignant B cells. In this study, the cytotoxic phenotype and function of various CD4+ T‐cell subgroups were investigated in patients with B‐cell malignancies. Peripheral blood was collected from patients with CLL, various B‐cell lymphomas, healthy adult donors, children with precursor B‐cell acute lymphoblastic leukaemia (pre‐B ALL) and from healthy children. CD4+ T cells (CD3+ CD4+ FoxP3−), Tregs (CD3+ CD4+ CD127low FoxP3+) and CD127high FoxP3+ T cells (CD3+ CD4+ CD127high FoxP3+) were analysed for their expression of the cytolytic markers CD107a and Fas ligand. Patients with CLL had increased CD107a expression on all tested T‐cell subgroups compared with healthy donors. Similar results were found in patients with B‐cell lymphomas whereas the CD107a expression in children with pre‐B ALL was no different from that in healthy controls. Fas ligand expression was similar between patient cells and cells of healthy donors. CD4+ T cells and Tregs from patients with CLL and healthy donors were subsequently purified and cultured in vitro with autologous B cells. Both subgroups lysed B cells and killing was confirmed by granzyme ELISAs. In conclusion, cytotoxic populations of CD4+ T cells, including Tregs, are present in patients with B‐cell malignancy and may be an important factor in immune‐related disease control.

Gamma-Aminobutyric Acid Concentration in Lumbar Cerebrospinal Fluid from Patients with Febrile Convulsions and Controls

ABSTRACT. The cerebrospinal fluid (CSF) concentration of the inhibitory neurotransmitter gamma‐aminobutyric acid (GABA) was analysed in 41 children with febrile convulsions (FC), 41 febrile controls of similar age (control group 1), and 59 controls, who had no fever and/or were outside the age range for FC (control group 2). A significant correlation between CSF‐GABA and age was demonstrated for controls (1 + 2) (r= 0.63, p < 0.00001), as well as for patients with FC (r= 0.42, p= 0.003). Patients with FC did not differ significantly from control group 1 in respect to CSF‐GABA. Duration of FC was related to both CSF‐GABA and age (GABA: r=−0.29, p < 0.05; age: r =−0.32, p < 0.05). For 56 controls (1 + 2) > 1 year of age, a significant negative correlation between CFC‐GABA and body temperature was found (r=−0.34, p = 0.01). The low CSF‐GABA in the FC‐labile age group, the negative correlation of CSF‐GABA to body temperature, and the negative correlation of the duration of FC to both CSF‐GABA and age, all indicate that GABA could be of importance in the pathophysiology of FC.

In vitro cellular drug sensitivity at diagnosis is correlated to minimal residual disease at end of induction therapy in childhood acute lymphoblastic leukemia

Leukemic cells from 85 children with newly diagnosed precursor B-lineage ALL were tested for in vitro drug sensitivity to a panel of anti-cancer drugs. Minimal residual disease (MRD) was measured by RQ-PCR. There was a significant correlation between MRD day 29 and in vitro sensitivity to prednisolone (p<0.001) and doxorubicin (p=0.017), drugs administered during induction therapy. In patients with t(12;21) (n=20), in vitro sensitivity to doxorubicin was an independent factor for MRD <0.1% (p=0.031; R(2)=0.66). Thus, data show that in vitro drug sensitivity at diagnosis is correlated to cell kill during induction therapy as measured by MRD day 29.

Applicability of IG/TCR gene rearrangements as targets for minimal residual disease assessment in a population-based cohort of Swedish childhood acute lymphoblastic leukaemia diagnosed 2002–2006

Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukaemia (ALL) treatment protocols. Here, we aimed to address the applicability of rearranged antigen‐receptor genes as potential MRD markers using real‐time quantitative polymerase chain reaction (RQ‐PCR) in a Swedish population‐based cohort. From 334 childhood ALL cases diagnosed during 2002–2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T‐cell receptor (TCR) genes. Allele‐specific oligonucleotides were designed, and the sensitivity and quantitative level was determined for each target. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B‐cell precursor ALL (BCP ALL) and 94% (33/35) of T‐ALL. A sensitive RQ‐PCR analysis (≤10−4) was obtained in 89% (216/244) of BCP ALL and in 74% (26/35) of T‐ALL, whereas two sensitive targets were only available in 47% (115/244) of BCP ALL and 29% (10/35) of T‐ALL cases. With the stratification threshold of ≥10−3, which is applied in the current Nordic treatment protocol (NOPHO‐ALL 2008) for the identification of high‐risk patients, 93% of BCP ALL and 86% of T‐ALL reached this quantitative range by at least one target gene. Taken together, this national retrospective study demonstrates that an IG/TCR target for MRD monitoring can be identified in the majority of childhood ALL cases, whereas identification of a second sensitive target gene needs to be improved.

Monitoring minimal residual disease with flow cytometry, antigen-receptor gene rearrangements and fusion transcript quantification in Philadelphia-positive childhood acute lymphoblastic leukemia

In this study, we followed minimal residual disease (MRD) in eight children with Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) using (i) flow cytometry (FCM), (ii) real-time quantitative PCR of IG/TCR gene rearrangements and (iii) RT-PCR detecting fusion gene transcripts. In six of the eight cases the kinetics of MRD clearance was comparable. One of the two discordant cases could be explained by presence of an alternative fusion transcript. The other discordant case showed high BCR-ABL1 RNA level while the other methods did not detect any MRD. In our limited material quantitative RT-PCR of fusion gene transcripts seemed particularly useful to measure MRD in Ph+ ALL. However, BCR-ABL1 expression may not reflect the percentage of leukemic cells as FCM and IG/TCR rearrangement quantification do, and these methods are thus complementary.

In vitro cellular drug resistance adds prognostic information to other known risk-factors in childhood acute lymphoblastic leukemia.

Leukemic cells from 230 children with newly diagnosed B-cell precursor ALL were tested for in vitro drug resistance to a panel of anti-cancer drugs. Minimal residual disease (MRD) was measured by RQ-PCR. During follow-up, 24 relapses occurred in the 159 children with MRD <0.1% day 29. The risk of any relapse was correlated to vincristine and doxorubicin resistance, with a relative risk of 3.7 (95% CI 1.3-10.5; p=0.016) for patients resistant to both drugs. There was a significant correlation also for the subgroup with extra-medullary relapses. Our findings indicate that analysis of drug resistance can add prognostic information to other known risk-factors including MRD.

U-2973, a novel B-cell line established from a patient with a mature B-cell leukemia displaying concurrent t(14;18) and MYC translocation to a non-IG gene partner

B‐cell lymphomas/leukemias with simultaneous t(14;18)(q32;q21) and MYC rearrangements have recently been shown to constitute a separate diagnostic entity, presenting with a rapid clinical course and a very poor prognosis. We describe the establishment of an Epstein–Barr virus negative cell line, designated U‐2973, from a male patient with a de novo aggressive B‐cell lymphoma/leukemia and very high peripheral blast cell count. Flow cytometry of bone marrow cells and U‐2973 displayed a mature B‐cell phenotype, and immunostaining showed expression of MYC and BCL2. IG gene rearrangement data were consistent with a lymphoid neoplasm of germinal centre derivation. Cytogenetic studies using conventional G‐banding, fluorescent in situ hybridization, spectral karyotyping and single nucleotide polymorphism array demonstrated a complex karyotype with both a t(14;18) and double translocations between MYC and a non‐IG gene partner located at chromosome 12p12.1.

Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

The ETV6/RUNX1 fusion transcript is not detected in RNA isolated from neonatal dried blood spots from children later diagnosed with the corresponding leukemia

The ETV6/RUNX1 fusion transcript is not detected in RNA isolated from neonatal dried blood spots from children later diagnosed with the corresponding leukemia