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A clinical course of patients with mantle cell lymphoma (MCL) is aggressive, and the disease is rarely curable. Proliferation rate is the most important prognostic factor. We developed a novel, reliable, rapid, and routinely applicable approach allowing a precise quantitative assessment of three proliferation markers, Ki-67, topoisomerase IIalpha, and TPX2. A total of 95 lymphoma specimens were measured in the study by real-time reverse transcription PCR (RQ-RT-PCR). We tested the reproducibility and accuracy of the assay and correlated the results with the immunohistochemical staining of the corresponding proteins. The results obtained indicated individual variability of the mRNA expression levels, reflecting heterogeneity of the proliferation rate in individual patients. In general, we observed the highest mRNA expression in the group of Burkitt lymphomas and the lowest in patients with reactive lymphadenopathies. We found increased proliferation rate in MCLs with high cyclin D1 mRNA, indicating a quantitative control of the cell cycle. We observed a correlation between mRNA expression level and the immunohistochemical staining of corresponding proteins, which significantly argues for the prognostic significance of the mRNA expression measuring. We confirmed the accuracy of the current assay for a precise quantitative examination of the proliferation activity. Real-time RT-PCR provides a novel approach applicable for clinical trials, and it represents a potent approach allowing to stratify MCL patients for entry into clinical trials according to the expression of the proliferation signature genes in their tumors. This approach may contribute to improved and individualized therapeutic options respecting the individual progression risk of patients with MCL.

In mantle cell lymphoma (MCL), minimal residual disease (MRD) is an indicator of the disease outcome. Quantitative methods used so far do not provide a suitable molecular marker in 30-70% patients with MCL (depending on the technique used). We tested cyclin D1 as a marker for quantitative MRD monitoring. The real-time PCR of cyclin D1 mRNA was performed in 144 bone marrow (BM) specimens including 95 BMs from MCL patients, 39 BMs from patients with other B-cell non-Hodgkin's lymphomas and 10 BMs from healthy volunteer donors. In 73 BMs obtained from 20 MCL patients we examined the cyclin D1 level during the treatment and follow-up period. We detected a cyclin D1 overexpression exclusively in BMs infiltrated with MCL, including minimal residual infiltration. Dynamics of cyclin D1 correlated with the patient's clinical status in 69/73 BMs. Individual monitoring of patients during the disease course showed cyclin D1 quantitative changes accompanying either the disease relapse or a successful treatment response or the disease-free survival (remission) and it showed a predictive significance. Cyclin D1 detection is a promising approach for the quantitative MRD monitoring in MCL patients, and the individual monitoring of the cyclin D1 dynamics represents a suitable indicator of the disease course., ((c) 2008 Wiley-Liss, Inc.)

Wilms' tumor gene 1 (WT1) is overexpressed in various hematological malignancies and has been proposed as a target for minimal residual disease (MRD) detection and for immunotherapy. Although WT1 is known as a key molecule for tumor cell proliferation, the expression pattern of WT1 in leukemic cells in dependency of proliferation has not yet been investigated. Furthermore, WT1 expression was mostly studied by reverse transcriptase PCR and the expression of WT1 protein has not been extensively studied. Here, we analyzed WT1 protein expression in the human myeloid leukemia cell lines K562 and HL-60 by indirect immunofluorescence and flow cytometry. Both cell lines exhibited varying nuclear WT1 immunoreactivity pointing to a cell cycle-dependent and/or proliferation-dependent WT1 expression. In rapidly proliferating cells high levels of WT1 protein were detected by flow cytometry. A reduced proliferation rate was associated with a low WT1 protein expression and an accumulation of cells in G(0)/G(1) phase. During G(0)/G(1) phase cells expressed WT1 at a lower level than in S or G(2)/M phase. Moreover, WT1 expression was diminished in all cell cycle phases in slowly proliferating cells. We conclude that WT1 protein expression is dependent on the cell cycle phase as well as on the proliferation rate. This finding might be relevant for MRD studies and immunotherapeutic strategies targeting WT1.

In patients with acute leukemia, Wilms' tumor gene 1 (WT1) has been used as a target for the detection of minimal residual disease (MRD) by PCR techniques. The expression of WT1 protein, however, has not been extensively studied. To determine the relation between expression of WT1 transcripts and of the encoded protein, we examined leukemic cell lines and primary childhood leukemia samples using both real-time quantitative PCR (RQ-PCR) and flow cytometry. WT1 protein was highly expressed in the leukemic cell lines K562, HL-60, PLB 985, KG-1a and CEM. By contrast, 40 primary samples of acute lymphoblastic leukemia (ALL; B-ALL, n = 15 and T-ALL, n = 10) and acute myeloid leukemia (n = 15) expressed low levels of WT1 protein. RQ-PCR detected WT1 transcript levels in the same range as reported in earlier studies in childhood acute leukemia. The results of this study indicate the following: (i) there are considerable discrepancies between WT1 transcripts and protein expression; (ii) WT1 is not a suitable marker for flow cytometric MRD detection in childhood acute leukemia., (Copyright 2008 Wiley-Liss, Inc.)

Cyclin D1 overexpression as a result of t(11;14) is a specific marker for diagnosis of mantle cell lymphoma (MCL) and plays an important role in MCL pathogenesis. To set a highly reliable cutoff value that discriminates MCL from other B-cell lymphoproliferative disorders, we performed a retrospective study of cyclin D1 expression. We established cyclin D1 expression level in 116 frozen and formalin-fixed, paraffin-embedded primary tumors from patients diagnosed with a variety of B-cell lymphoproliferative disorders. We used real time quantitative reverse-transcription polymerase chain reaction to quantify levels of cyclin D1 mRNA. The range of cyclin D1 expression in MCLs exceeded the range found in other lymphomas and reactive lymph nodes by a considerable margin. Cyclin D1 overexpression was found in 60/61 MCLs and in none of the other lymphomas, except for 12/19 mucosa-associated lymphoid tissue lymphomas from the lungs and stomach, which also revealed cyclin D1 overexpression. As epithelial tissues are known to express cyclin D1, an admixture of non-neoplastic epithelial cells present in the extranodal specimens probably influenced the quantitative reverse-transcription polymerase chain reaction result. Quantitative cyclin D1 monitoring provides a diagnostic test and an approach for studying MCL pathogenesis and may be of clinical importance.

Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of malignant lymphoproliferative diseases with a consistent expression of the cytokine receptor CD30. ALCL is frequently associated with a NPM/ALK fusion gene which is found in up to 75% of pediatric ALCLs. Real-time quantitative RT-PCR (RQ-RT-PCR) of NPM/ALK and CD30 gene expression was employed to analyze minimal residual disease (MRD) in 10 patients with NPM/ALK positive ALCL in 79 follow-up bone marrow (BM) and/or peripheral blood (PB) samples. In all BM samples from relapses and/or closely before a relapse, BM samples revealed NPM/ALK and CD30 positivity in at least one of the iliac BM trephines. Five out of nine relapses were preceded or were accompanied by minimally half log increased NPM/ALK levels in the BM. We found that RQ-RT-PCR of the CD30 expression is not suitable for MRD detection--only two relapses were accompanied by an increase of the CD30 level above a level which was detected in BM/PB samples from healthy individuals. RQ-RT-PCR of NPM/ALK expression is a promising and rapid approach for monitoring MRD.

Background: Lung carcinogenesis is a multistep process of accumulation of genetic changes, including loss of heterozygosity (LOH), and precedes phenotypic transformation of the bronchial mucosa. The activity of telomerase, correlating with the hTERT mRNA expression, is detectable in a majority of neoplasms. In this study, the frequency of LOH and hTERT expression in bronchial mucosa of heavy smokers in bronchoscopic biopsies was analyzed., Methods: LOH was examined in 122 bronchial specimens from 81 smokers (67 normal mucosa/bronchitis, 12 squamous metaplasia, 28 dysplasia, 15 bronchogenic carcinoma specimens) by polymerase chain reaction (PCR) and capillary electrophoresis by using 7 fluorescence-labeled markers matching 5 chromosomal regions. hTERT expression was analyzed in 87 specimens (45 normal mucosa/bronchitis, 12 squamous metaplasia, 18 dysplasia, 12 bronchogenic carcinoma specimens) by real-time quantitative reverse-transcription PCR., Results: LOH was detected in at least 1 chromosomal region in 51 of 122 (41.8%) specimens; the incidence in normal bronchial mucosa and preneoplastic lesions was similar (20%-40%); a substantial rise (87%) occurred in carcinomas. The median normalized hTERT(N) values were 6.67 in normal epithelium/chronic bronchitis, 18.38 in squamous metaplasia, 13.31 in epithelial dysplasia, and 75.46 in carcinomas. These results were significantly different (P=.0036). With an increasing number of LOH, the median value of hTERT(N) expression rose, but hTERT was expressed also in tissue samples without any LOH detection., Conclusions: Results indicated that hTERT expression, together with LOH, represent early events in lung carcinogenesis, as both were detected in precancerous lesions and in normal epithelium of heavy smokers.

Gene expression profiles of 10 children with acute lymphoblastic leukemia (ALL) were detected using cDNA arrays. Total RNAs were isolated from peripheral blood leukocytes of the patients at diagnosis. For detection of expression profiles we used Atlas Human Cancer cDNA Arrays (Clontech) with 588 genes. Although the study revealed variability of gene expression in many genes, we identified a number of genes with the same expression changes (up-regulation: PCNA, ERCC1; down-regulation: jun-B, BCL-2 related protein A1, CRAF-1, PBP) in most examined patients. Our objective was to identify genes that were differentially expressed in ALL and might contribute to development (and characterization) of the disease.