Anna Porwit-MacDonald

Microarray-based classification of a consecutive series of 121 childhood acute leukemias: prediction of leukemic and genetic subtype as well as of minimal residual disease status.

Gene expression analyses were performed on 121 consecutive childhood leukemias (87 B-lineage acute lymphoblastic leukemias (ALLs), 11 T-cell ALLs and 23 acute myeloid leukemias (AMLs)), investigated during an 8-year period at a single center. The supervised learning algorithm k-nearest neighbor was utilized to build gene expression predictors that could classify the ALLs/AMLs according to clinically important subtypes with high accuracy. Validation experiments in an independent data set verified the high prediction accuracies of our classifiers. B-lineage ALLs with uncharacteristic cytogenetic aberrations or with a normal karyotype displayed heterogeneous gene expression profiles, resulting in low prediction accuracies. Minimal residual disease status (MRD) in T-cell ALLs with a high (>0.1%) MRD at day 29 could be classified with 100% accuracy already at the time of diagnosis. In pediatric leukemias with uncharacteristic cytogenetic aberrations or with a normal karyotype, unsupervised analysis identified two novel subgroups: one consisting mainly of cases remaining in complete remission (CR) and one containing a few patients in CR and all but one of the patients who relapsed. This study of a consecutive series of childhood leukemias confirms and extends further previous reports demonstrating that global gene expression profiling provides a valuable tool for genetic and clinical classification of childhood leukemias.

BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation.

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.

Flow cytometric follow-up of minimal residual disease in bone marrow gives prognostic information in children with acute lymphoblastic leukemia

Using flow cytometry (FC) and live gate (LG) analysis we have followed levels of minimal residual disease (MRD) in the bone marrow (BM) of 70 consecutive patients with childhood acute lymphoblastic leukemia (59 B precursor ALL and 11 T-ALL) treated according to the Nordic (NOPHO-92) protocols. Thorough studies of B and T cell antigen expression patterns in normal BM performed during BIOMED 1 Concerted Action on MRD, made it possible to tailor individual protocols of marker combinations for follow-up in 97% of patients. In 12% of LG analyses, the numbers of cells exceeded 106 and in 82% exceeded 105, giving the sensitivity level of MRD detection 10−5 and 10−4, respectively. The median follow-up time was 53 months. Patients with MRD levels ≥0.01% at follow-up time-points during and after first induction, and at the end of treatment had significantly lower disease-free survival by comparison to patients with MRD values <0.01%. Seven of nine patient with recurrence in the BM showed under treatment persisting MRD levels ≥0.01% of BM cells. This was also observed in another two patients with infant leukemia who relapsed. In conclusion, the investigation of levels and the dynamics of MRD by sensitive and quantitative FC can provide a basis for further clinical studies for at least upgrading of therapy.

Analysis of minimal residual disease in childhood acute lymphoblastic leukemia: comparison between RQ-PCR analysis of Ig/TcR gene rearrangements and multicolor flow cytometric immunophenotyping.

Detection of minimal residual disease (MRD) in follow-up samples from patients with ALL is essential for evaluation of treatment response. We applied multicolor flow cytometry and real-time quantitative PCR (RQ-PCR) to compare MRD results in 71 follow-up samples from 22 children treated for ALL. When results obtained by flow cytometry and RQ-PCR were grouped into positive–negative categories, a significant level of agreement was found in 72% of samples (P<0.001). However, if a cutoff level of 0.01% was applied, the concordance was 89%. MRD could be quantified in 19 samples by both methods, showing a strong correlation (P<0.01). Nevertheless, MRD levels differed more than five-fold between both methods in 4/19 samples. In 20 (28%) samples, the two techniques showed discordant results. Most discordant results (17/20) were due to the limited sensitivity of flow cytometry analysis within the range 0.01–0.001%; remaining discordant results were due to the instable or subclonal IG/TCR gene rearrangements or a limited quantitative range of the applied RQ-PCR targets. Although concordant results could be obtained by flow cytometry and RQ-PCR analysis, MRD levels may differ. Therefore, MRD data obtained by these two techniques are not yet easily exchangeable.

Flow Cytometric Immunophenotyping Including Bcl-2 Detection on Fine Needle Aspirates in the Diagnosis of Reactive Lymphadenopathy and Non-Hodgkin's Lymphoma

Fine‐needle aspiration (FNA) with immunophenotyping by immunocytochemistry (IC) on cytospins has recently received increased consideration in the diagnosis of lymphoma. The aim of our study was to establish the diagnostic value of a four‐color flow cytometric (FCM) panel, including cytoplasmic Bcl‐2, in cytologic diagnosis of malignant non‐Hodgkins lymphoma (NHL) and reactive lymphoid hyperplasia (RH).

Flow cytometry and allele-specific oligonucleotide PCR are equally effective in detection of minimal residual disease in ALL.

The analysis of minimal residual disease (MRD) has assumed a growing role in the follow-up of patients with acute lymphoblastic leukemia (ALL). We have applied multiparameter flow cytometry (FC) with ‘live-gate’ analysis and allele-specific oligonucleotide (ASO)-PCR detecting leukemia-specific T cell receptor γ and δ gene rearrangements for MRD follow-up in 30 ALL patients. The comparison of results obtained in 89 follow-up samples from 23 patients showed significantly consistent results in 70 samples (78%); (P < 0.001). Bone marrow samples taken during the first phase of treatment (during or immediately after induction) showed a lower level of consistency when compared to samples taken during later phases of treatment (69% vs 85% consistent results, respectively). Some of the discrepant results were due to low cellularity of the samples obtained for FC and some due to the presence of PCR inhibitors. Of 29 patients evaluated at the end of the induction treatment, 18 (62%) had detectable levels of MRD and six of these patients suffered relapse. In all these patients MRD levels by FC increased preceding relapse. Our results suggest that FC offers a MRD detection tool that can be easily applied in clinical practice and is as informative as molecular methods.

Mantle cell lymphomas acquire increased expression of CCL4, CCL5 and 4-1BB-L implicated in cell survival.

We have analyzed mantle cell lymphomas (MCLs), using high‐density DNA microarrays, and confirmed the expression of differentially regulated antigens, using flow cytometry and immunohistochemistry. The results show that MCLs acquire expression of molecules that normally are involved in interaction with other immune cells and, thus, might affect the ability of the tumor to survive. The MCL signature is represented by the overexpression of the chemokine CCL4 (MIP‐1β), implicated in the recruitment of regulatory T cells, as well as CCL5 and 4‐1BB‐L. The latter molecules are normally involved in chemotaxis of T cells and B cell activation, respectively. Signaling through 4‐1BB‐L allows B cells to proliferate and the expression of its ligand, by the intra‐tumoral mesh of follicular dendritic cells (FDC), could thus serve as a paracrine loop facilitating growth and survival of MCL cells.

Mantle cell lymphomas with low levels of cyclin D1 long mRNA transcripts are highly proliferative and can be discriminated by elevated cyclin A2 and cyclin B1.

The role of transcript variants of cyclin D1 in cancer biology is unclear. Most tumors with high levels of cyclin D1 express 2 transcripts due to alternative splicing: one full‐length transcript of 4.4 kb and one short transcript of approximately 1.7 kb. The short transcript lacks part of the 3′UTR region regulating mRNA stability and has a longer half‐life. In our study, the contribution of each of these mRNAs to gene expression and cell proliferation has been investigated in mantle cell lymphoma (MCL), a B cell lymphoma characterized by a specific gene translocation resulting in enhanced expression of cyclin D1. A subset of MCL tumors with low levels of the long cyclin D1 transcript (cyclin D1 3′UTR) was identified by quantitative PCR and by oligonucleotide array hybridization. This tumor‐subset had 3.4‐fold higher levels of the short form of cyclin D1 mRNA (p < 0.0001) and had higher expression of cyclin D1 protein. Gene expression analysis identified a number of cell‐cycle regulatory genes as upregulated. There was a significant difference in frequencies of cyclin B1 (p = 0.0006) and cyclin A2 (p = 0.0006) positive cells that discriminated MCL with low cyclin D1 3′UTR from other highly proliferative MCL. Among differentially expressed genes, there was a highly upregulated gene with homology to the group of cell‐cycle promoting E2F transcription partners, E2F_TDP5. Several of the upregulated genes, such as TOP2A, AURORA A and RRM2 may influence a response to therapy. Identification of MCL with low cyclin D1 3′UTR is important because it seems to be associated with shorter overall survival.

Cigarette Smoking and Risk of Non-Hodgkin's Lymphoma—A Population-Based Case-Control Study

Background: Epidemiologic evidence of an association between tobacco smoking and non-Hodgkins lymphoma has been conflicting. This may reflect that non-Hodgkins lymphoma comprises several distinct disease entities with different etiologies, as some studies have indicated an association between smoking and follicular lymphoma. Objective: To investigate the association between cigarette smoking and non-Hodgkins lymphoma risk, overall and by subtype. Methods: As part of a nationwide Danish-Swedish population-based case-control study, we interviewed 3,055 incident non-Hodgkins lymphoma patients and 3,187 population controls. All lymphomas were uniformly classified according to the WHO classification. We used unconditional logistic regression to estimate adjusted odds ratios (OR) and 95% confidence intervals (95% CI) for the association between cigarette smoking and risk of non-Hodgkins lymphoma. Results: Cigarette smoking was not associated with the risk of non-Hodgkins lymphoma overall (OR, 0.97; 95% CI, 0.87-1.08) nor with the major subgroups such as diffuse large B-cell lymphoma (OR, 0.94; 95% CI, 0.79-1.10), chronic lymphocytic leukemia (OR, 0.86; 95% CI, 0.72-1.02), or follicular lymphoma (OR, 1.03; 95% CI, 0.85-1.24). Female smokers were at a marginally increased risk of follicular lymphoma (OR, 1.41; 95% CI, 1.04-1.92). Men who had ever smoked had a significantly increased risk of T-cell lymphoma (OR, 1.67; 95% CI, 1.11-2.51). No dose-response association with cigarette smoking could be established for any lymphoma subgroup. Conclusion: We found little evidence of an association between cigarette smoking and non-Hodgkins lymphoma risk overall. Although increased risks of follicular lymphoma in female smokers and of T-cell lymphoma in male smokers were suggested, no dose-response relationship was observed, leaving limited support for causality.

Expression of LAZ3/BCL6 in follicular center (FC) B cells of reactive lymph nodes and FC-derived non-Hodgkin lymphomas

Chromosomal translocation resulting in abnormal expression of the LAZ3/BCL6 gene in B cells has been implicated in the tumorigenesis of non-Hodgkin lymphoma (NHL). Therefore we studied the expression pattern of LAZ3/BCL6 by in situ hybridization with synthetic oligonucleotide probes in frozen tissue sections from five reactive lymph nodes and 38 B cell and non-B NHL. In addition, we investigated the expression of LAZ3/BCL6 by Northern blot analysis on multiple human tissues. The LAZ3/BCL6 transcript was found in a variety of tissues, including skeletal muscle, peripheral blood leukocytes, and weakly in normal lymph nodes. In the tumor samples, expression of LAZ3/BCL6 was observed in 68% of all B cell NHL and none of the non-B lymphomas. All cases of follicular, mixed small and large cell lymphomas showed LAZ3/BCL6 expression confined to the neoplastic follicles. A follicular expression pattern was also found in all non-malignant reactive lymph nodes. Hence, the expression of LAZ3/BCL6 does not correlate to malignancy, but reflects the origin of B cells from the germinal centers.