hTERT, the catalytic component of telomerase, is downregulated in the haematopoietic stem cells of patients with chronic myeloid leukaemia.

Telomere shortening is associated with disease progression in chronic myeloid leukaemia (CML). To investigate the biology and regulation of telomerase in CML, we evaluated expression of the telomerase components, its regulators and several telomeric-associated proteins. Quantitative real-time-polymerase chain reaction (PCR) was used to compare gene expression in the CD34+/leukaemic blast cells of 22 CML patient samples to the CD34+ cell population of healthy individuals. hTERT, the catalytic component of telomerase, was downregulated in eight of 12 chronic phase (CP) patients (P = 0.0387). Furthermore, hTERT was significantly downregulated in two of three patients in accelerated phase (AP) and seven of seven patients in blast crisis (BC), P = 0.0017. Expression of hTR and telomeric-associated proteins TEP1, TRF1, TRF2, tankyrase and PinX1 was high in the majority of CP and AP patients. With the exceptions of TEP1 and hTR, expression of these factors was highest in CP and decreased during disease progression. Expression of c-Myc, a positive regulator of hTERT transcription, correlated with hTERT expression and decreased with disease progression, falling below control levels in BC. hTERT levels were increased in CP patients following successful treatment with imatinib, relative to untreated CP patients. We suggest that reduced hTERT expression directly causes the shortened telomeres observed in CML.

Templated nucleotide addition and immunoglobulin JH-gene utilization in t(11;14) junctions: implications for the mechanism of translocation and the origin of mantle cell lymphoma.

The t(11;14)(q13;q32) between the BCL-1 and immunoglobulin heavy chain gene (IgH) loci in mantle cell lymphoma (MCL) are believed to be mediated by the mechanism of V(D)J recombination similar to the t(14; 18) in follicular lymphoma (FL). We have recently shown that the t(14;18) event creates staggered double-strand breaks in the BCL-2 locus, and that the t(14;18) junctions contain templated nucleotide insertions (T-nucleotides; U. Jäger et al., Blood, 95: 3520-3529, 2000). Reasoning that the earlier (pregerminal center) B-cell origin of MCL might be reflected in a different molecular structure of the chromosomal breakpoints, we PCR-amplified diagnostic samples from 93 patients. Thirty-six samples (39%) were positive for the direct (BCL-1/J(H)) and 23 for both direct and reciprocal (D(H)/BCL-1) junctions. The breaks on chromosome 14 exhibited features of V(D)J-mediated recombination as shown by D(H) and J(H) coding end processing. However, duplications of BCL-1 sequences in 39% of the 23 patients indicate staggered double-strand breaks in the major translocation cluster region (MTC). This is incompatible with V(D)J recombination and indicates a different mechanism of cleavage. The use of J(H)6 in the junctions (39%) was similar to that in the immunoglobulin genes of normal B cells and B-CLL, but considerably less than in FL. Only 2 of 36 samples contained a BCL-1/DJ(H) rearrangement, which was indicative of a previous DJ(H) rearrangement. Most importantly, 19% of the BCL-1/IgH junctions with inserts of > or =5 nucleotides contained error-prone copies (T-nucleotides) of 8-12 nucleotides originating from the surrounding BCL-1 or IgH regions, a lower rate than in FL. No correlation was found between the addition of T-nucleotides and the rate of somatic mutation in the immunoglobulin genes. We conclude that the t(11;14) and t(14;18) use the same basic mechanism of translocation including V(D)J-mediated recombination, double-strand staggered breaks, and template-dependent, error-prone DNA-synthesis. However, the distinct differences in the utilization of J(H) regions suggest that the t(11;14) occurs predominantly during an attempted primary D(H)-J(H) rearrangement in early B cells, whereas the t(14;18) mostly occurs during secondary rearrangement. This is in agreement with the pregerminal center B-cell origin of MCL.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes.

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines.

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease.

AML1/ETO fusion mRNA can be detected in remission blood samples of all patients with t(8;21) acute myeloid leukemia after chemotherapy or autologous bone marrow transplantation.

The chromosomal translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) can be detected by a reverse transcription-polymerase chain reaction (RT-PCR) for the chimeric AML1/ETO transcript. We have evaluated the clinical relevance of this method for monitoring and detection of minimal residual disease (MRD) in seven patients who reached a complete hematological remission (CHR) after chemotherapy or autologous bone marrow transplantation (ABMT). Peripheral blood (PB) samples of five patients in first continuous complete remission (CCR) were still PCR-positive at a frequency of 1 in 10(5) cells after 7, 8, 8, 10 or 66 months. Chemotherapy led to a reduction from first- to second-step PCR-positivity in three serially monitored patients. AML1/ETO mRNA was also detected in the PB of two patients in CCR, 10 or 12 months after ABMT. PB and bone marrow (BM) showed identical results in all samples tested simultaneously. AML1/ETO fusion transcripts were neither found in the PB and BM of a healthy individual, nor in the PB of a patient after allogeneic BMT for cytogenetically proven t(8;21)-leukemia. Our results indicate the presence of cells carrying the AML1/ETO rearrangement in the PB and BM of all patients in CHR after chemotherapy or ABMT for t(8;21)-positive AML. While this finding raises interesting questions about the biology of acute leukemia, it limits the value of the AML/ETO RT-PCR for the prediction of impending relapse.

Prognostic significance of WT1 gene expression at diagnosis in adult de novo acute myeloid leukemia.

We examined the presence of WT1-specific mRNA in bone marrow samples of 125 patients with de novo acute myeloid leukemia at diagnosis by two-step RT-PCR. The sensitivity of the assay was 1:100 (first step) and 1:10000 (second step), respectively. WT1-specific mRNA was detected in 73% of patients. No correlation was found between WT1 gene expression and age, FAB type, LDH and karyotype at diagnosis. All patients were treated with standard induction chemotherapy. There was no difference in the CR rate between WT1-positive and -negative patients. Using Kaplan and Meier plot analysis we found no difference in disease-free survival (DFS) and overall survival (OS) between patients displaying the WT1 transcript and WT1-negative patients. Furthermore, no significant interactions between WT1 PCR results and age, FAB type, LDH and karyotype on DFS and OS were demonstrable using Cox regression analysis. Eight patients who were WT1 PCR positive at diagnosis and achieved complete hematological remission following chemotherapy were monitored during the course of the disease. Based on our limited data demonstrating a heterogeneity of WT1 PCR results in CR we cannot draw any conclusions regarding the usefulness of WT1 PCR analysis for the early detection of relapse. We conclude that WT1 gene expression at diagnosis is not associated with specific characteristics of AML blast cells and is not a prognostic factor for CR, remission duration and overall survival in acute myeloid leukemia.

Molecular mechanism of the t(14;18)--a model for lymphoid-specific chromosomal translocations.

The chromosomal translocation t(14;18) occurs during early B-cell development and juxtaposes the immunoglobulin heavy chain locus (IgH) with the bcl-2 oncogene. Several factors contribute to the translocation mechanism: (1) The rearrangement of the chromosome 14 DH and JH translocation partners has typical features of V(D)J-recombinase-mediated joining with N-segment addition. (2) The bcl-2 major (mbr) and minor (mcr) breakpoint regions as well as their IgH reciprocal counterparts contain recombinatorial sequences related to chi or the minisatellite-core which bind at least one common DNA-binding protein (bp45). Similar elements are found at the breakpoints of other lymphoid-specific translocations like the t(11;14), t(2;8) or the t(4;11). (3) Structural analysis of the bcl-2 mbr indicates that this region may adopt alternative DNA-configurations which can promote recombination and is cleaved by an endogenous nuclease present in early B-cells. The present data suggest that V(D)J-recombinase as well as chi/minisatellite-core mediated recombination contribute to the mechanism and make the t(14;18) a model system for lymphoid-specific reciprocal translocations.

A pilot study of mitochondrial DNA point mutation A3243G in a sample of Croatian patients having type 2 diabetes mellitus associated with maternal inheritance.

In this work, patients having type 2 diabetes mellitus and diabetic mothers were tested for the presence of mitochondrial DNA point mutation A3243G. This mutation is associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), diabetes and deafness. Twenty-two diabetic persons were screened. DNA was isolated from peripheral blood lymphocytes and from swabs of oral mucosa. The mitochondrial DNA point mutation A3243G was detected using PCR-RFLP test. The mutation was detected in oral mucosal DNA of two patients (but not from lymphocyte DNA). One patient was a man with hearing and visual impairments and proteinuria; the other was a woman having proteinuria but no hearing impairment. The mutation was not detectable in oral mucosal DNA from the control persons: 20 diabetic patients having diabetic fathers and 22 healthy, nondiabetic volunteers. The incidence of mitochondrial DNA point mutation A3243G in this study of Croatian diabetic patients is in line with data in the literature.

Reverse transcriptase-polymerase chain reaction (rt-PCR).

Reverse transcriptase polymerase chain reaction (RT-PCR) has become a well-established and powerful molecular technique for studying ribonucleic acids. It is used in medical diagnostics for detecting viral RNA, in hematology and oncology for detecting chimeric transcripts of rearranged genes (1), and in the broad area of research applications in gene expression studies. Since its introduction (2,3) the method is constantly finding new applications, especially in projects aimed at detecting and cloning differentially expressed genes (4,5).

High-density single nucleotide polymorphism array analysis and ASXL1 gene mutation screening in chronic myeloid leukemia during disease progression.

We have undertaken a genome-wide single nucleotide polymorphism (SNP) array analysis of 41 chronic myeloid leukemia (CML) patients. In total, 44 regions of uniparental disomy (UPD) >3 Mb were identified in 24 of 32 patients in chronic phase (CP), and 21 regions of UPD >3 Mb were identified in 13 of 21 patients in blast crisis (BC). Chromosome 8 had the highest frequency of UPD regions in both CP and BC samples. Eight recurrent regions of UPD were observed among the 41 patients, with chromosome 8 showing the highest frequency. Ten regions of copy number change (CNC) >3 Mb were observed in 4 of 21 patients in BC, whereas none were observed in CP. We have identified several recurrent regions of UPD and CNC in CML that may be of pathogenetic importance. Overrepresentation of genomic aberrations (UPD and copy number gain) mapping to chromosome 8 was observed. Selected candidate genes mapping within the aberrant genomic regions were sequenced and mutation of the TP53 gene was observed in one case in BC and of the ASXL1 gene in 6 of 41 cases in CP or BC. Mutation of ASXL1 represents an important new molecular abnormality in CML.

Myeloid hemopoietic growth factors. Review article.

The article is a critical review of recent publications on myeloid hemopoietic growth factors. They are glycoproteins that regulate the proliferation and differentiation of hemopoietic progenitor cells and the function of mature blood cells. They are also named colony stimulating factors after the experimental technique that led to their discovery. Myeloid growth factors are: granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF) and interleukin-3. Studies of hemopoietic growth factors in the past years have revealed many potential beneficial effects, but some limitations as well. They have been proved effective, above all, in neutropenic conditions of different origin. A large number of clinical trials have shown their beneficial effect in myelosuppressive conditions after cytotoxic chemotherapy, in post-transplant period in bone marrow transplantation procedures and in neutropenic conditions as part of other clinical entities. Although a number of studies that should determine their clinical role more clearly is still underway, it seems certain that myeloid hemopoietic growth factors already have an important role in modern pharmacotherapy.

The roles of age and sex in the prognosis of chronic leukaemias. A study of 373 cases.

The roles of age and sex and their relationship to other prognostic factors were studied in 117 chronic myeloid leukaemia (CML) and in 256 chronic lymphocytic leukaemia (CLL) patients. Survival in CML was not related either to age at diagnosis or to sex. In contrast, the CLL patients classified into four age strata (less than 50, 50-59, 60-69, greater than 70 years) had an expected median survival (EMS) of 142, 101, 85 and 33 months respectively (chi 2 for heterogeneity = 35.59, P less than 0.0005; chi 2 for trend = 25.09, P less than 0.0005). Prognostic power was independent of sex, Rai stages, total tumour mass score (TTM), TTM distribution pattern, anaemia, thrombocytopenia, serum immunoglobulins and response to therapy. The relative survival rate (the ratio of patient's EMS and EMS in age- and sex-matched general population) was 0.40 in CLL patients and 0.13 in CML patients. Relative survival was more reduced in older CLL patients than in younger ones (0.37 vs 0.47, respectively), whereas relative survival was less reduced in older CML patients than in younger ones (0.18 vs 0.12, respectively). The results show that the age is a significant independent prognostic factor in CLL but not in CML. The difference in the effects of age on prognosis in CLL and CML most probably reflects the fundamental differences in their respective pathogeneses.

Assessment of Prognostic Factors in 102 Patients with Myelodysplastic Syndrome-Osteoblasts are Related to Survival.

In a group of 102 patients with myelodysplastic syndrome an evaluation of bone marrow biopsy was performed in order to assess prognostic impact on survival. The following characteristics were included in the analysis: cellularity, erythropoiesis, granulopoiesis, thrombopoiesis, lymphatic nodules, mastocytes, dyserythropoiesis, dysgranulopoiesis, dysthrombopoiesis, blasts, atypical localisation of immature precursor cells, fibrosis, osteoblasts and osteoclasts on bone surfaces. Data were statistically analysed by stepwise Cox proportional hazards regression. Survival was compared between FAB sub-classes, with better prognosis for RA and RARS patients and poor for CMML patients. Better survival was found in patients without dysgranulopoiesis and in those with osteoblasts on bone surfaces. Agreement with established prognostic factors indicates a representative sample. Relationship of osteoblasts to better survival shows involvement of bone tissue in MDS, suggesting it might be useful to include assessment of bone tissue features in histopathologic evaluation.

Comparison of karyotype analysis and RT-PCR for AML1/ETO in 204 unselected patients with AML.

The chromosomal translocation t(8;21) (q22;q22) is often associated with acute myeloid leukemia with maturation (AML-M2) and can be detected by a reverse transcription-polymerase chain reaction (RT-PCR) for the AML1/ETO fusion mRNA. We investigated the prevalence of t(8;21) and AML1/ETO in 204 unselected patients with AML and compared the results of cytogenetic analysis with these of RT-PCR. Fifteen of 204 AML patients (7.4%) showed a t(8;21) in karyotype analysis. In 17 of 204 patients (8.3%) AML1/ETO was detected by RT-PCR. All patients who had a t(8;21) in conventional karyotyping also showed the gene rearrangement in molecular analysis, including one patient with a three-way translocation t(5;8;21). AML1/ETO was also detected in two AML patients lacking the t(8;21) cytogenetically. One had a normal diploid karyotype bone marrow (BM) at diagnosis; she has now been in CCR for 12 months. The second patient showed a complex chromosomal anomaly involving chromosome 21, but without a typical 8;21 translocation (BM in relapse). He died in relapse after an overall survival of 60 months. These data indicate that the results of karyotyping and RT-PCR are not completely identical, and molecular biology identifies approximately an additional 5-10% of AML1/ETO positive cases. The clinical relevance of our findings will have to be evaluated with larger patient numbers.

The relevance of multidrug resistance-associated P-glycoprotein expression in the treatment response of B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine whether expression of P-glycoprotein (Pgp) is an intrinsic feature of B-lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) and how it correlates with hematologic indices and tumor load in the disease. Furthermore, the change of Pgp expression under cytotoxic treatment and its correlation to treatment outcome were studied. DESIGN AND METHODS: In 42 B-CLL patients, of whom 13 were sequentially monitored, expression of extracellular (MRK-16) and intracellular (C-219) Pgp epitopes on peripheral blood lymphocytes was determined by flow cytometry analysis and quantified by ratio of the mean fluorescence (RMF) in flow cytometry analysis. RESULTS: Median RMF values in B-CLL patients were higher than in age-matched controls. Pgp expression did not correlate with any of the hematologic features or clinical stage of the disease. Patients who received some type of cytoreductive treatment prior to the study had lower Pgp values for both measured epitopes (median RMF for C-219 and MRK-16 of 1.99 and 2.03 in comparison to those of non-treated patients: 3.11 and 2.88, respectively). In 13 patients monitored during treatment the decrease in RMF was noted after treatment with chlorambucil, with RMF values for both Pgp epitopes decreasing in responders. This was in contrast to unchanged or even increased RMF values in those patients who did not respond to therapy. INTERPRETATION AND CONCLUSIONS: Our study confirms the importance of quantitative evaluation of Pgp expression by flow cytometry. At the clinical level, cross-sectional, single test evaluation of Pgp is of limited value whereas sequential follow-up values correlate with treatment response.

Prolegomenon for chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40; thereafter the incidence of CLL increases exponentially with age. CLL is characterized by progressive expansion of malignant CD5+ME+ B-cell clone accompanied by a myriad of cellular and humoral immune defects. Each of them might be linked to different clinically manifested complications such as increasing rate of infections, autoimmune disorders and disturbed immune surveillance against tumour cells. We assume that CLL occurs as a consequence of age-dependent, genetically related functional restrictions of the thymic microenvironment in supporting common lymphoid progenitor cells (CD5+ME+CD4-CD8-) to differentiate into mature T-cell and B-cell descendants. In conjunction with genetic abnormalities developing in B-cell progenitors, presumably expressing P glycoprotein (Pgp+), we postulate that developmentally altered T-cell descendants, along with quantitative imbalance among CD4+, their subsets and CD8+ lymphocytes in the peripheral blood, play an important additional role in facilitating the malignant B-cell clone emergence and in modulating the CLL clinical evolution. Namely, imbalance of any of T-cell-mediated cell interactive homeostatic mechanisms accompanied by imbalance in the production of various cytokines might in CLL influence leukaemic B-cell growth by deregulating inducer (c-myc and p53) and/or suppressor (bcl-2 and mutant p53) oncogenes responsible for the promotion or suppression of B-cell mitogenesis that may in turn further contribute to their impaired differentiation and/or differentiation arrest. In conclusion, CLL might be interpreted as a primary immunodeficiency syndrome developing in elderly population due to gradually evolving restriction of genetically controlled programs in the thymic microenvironment responsible for irregular maturation of common lymphoid progenitor cells that constitutively express CD5 antigen and ME receptor into T-cell and B-cell descendants.

Treatment of chronic lymphocytic leukemia in early and stable phase of the disease: long-term results of a randomized trial.

In 1982 the IGCI CLL cooperative group decided to investigate the usefulness of treating, at diagnosis B-cell chronic lymphocytic leukemia (CLL) in early and stable phase of the disease. From January 1982 to December 1986, 148 patients were randomized either to receive immediate treatment with chlorambucil (CLB) or to defer therapy to the time of progression. The early and stable phase of the disease was defined by a total tumor mass (TTM) score < 9, the absence of anemia or thrombocytopenia and a doubling time > 12 months. The main end-point of the study was survival. At the last evaluation in April 1993, after a median follow-up of 75 months, no significant difference was found in overall survival between early vs. deferred treatment patients from every cause of death as well as from death due to CLL-related causes only. The same results were obtained when the patients in more favorable stages, such as Binet stage A and TTM < 4.5, were considered. Interestingly, the incidence of epithelial cancer was similar in the two groups. Early treatment was associated with a significantly better response and a lower progression rate. From this long-term experience, it can be concluded that immediate chemotherapy with CLB is not beneficial for CLL patients in early and stable phase of the disease in terms of survival.

Primary non-Hodgkin lymphoma of the humerus following traumatic injury: case report.

A case of primary non-Hodgkin lymphoma of the right humerus which occurred in a 21-year-old male patient after an impact to the right shoulder in a car accident in July 1983 is described. Seventeen years after the injury, due to a civil lawsuit, the biopsy material was revised. Immunohistochemical analysis showed CD20 and CD79a positivity on large pleomorphic cells, while small reactive lymphocytes were CD3, Bcl-2 and CD20 positive. Molecular analysis carried out with PCR revealed a monoclonal B-lymphocyte population. The diagnosis of diffuse large peripheral B cell lymphoma of the bone was confirmed. The present case concurs with the literature on primary bone lymphoma, in which the diagnostic problem, trauma-related presentation and an excellent prognosis of malignant tumour are emphasized.

High dose chlorambucil versus Binet's modified cyclophosphamide, doxorubicin, vincristine, and prednisone regimen in the treatment of patients with advanced B-cell chronic lymphocytic leukemia. Results of an international multicenter randomized trial. International Society for Chemo-Immunotherapy, Vienna.

BACKGROUND: In recent years, much attention has been paid to the possible efficacy of intensive chemotherapy in the treatment of advanced, progressive B-cell chronic lymphocytic leukemia (CLL) patients. For this reason, the International Society for Chemo-Immunotherapy, Chronic Lymphocytic Leukemia Cooperative Group, has begun a randomized multicenter trial comparing Binet's modified cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen with continuous high dose chlorambucil (HD-CLB). METHODS: During the period January 1987 to May 1993, 228 previously untreated CLL patients from 7 cooperative institutions were randomized to this trial. Advanced and/or progressive disease was defined by high Total Tumor Mass (TTM) score (> 9), and/or short doubling time (DT) (< 12 months), and/or bone marrow failure. The response to therapy was defined by reduction of the initial TTM score. The end points of the trial were response rate, survival, and toxicity. RESULTS: HD-CLB resulted in a higher response rate than CHOP in evaluable cases, with 89.5% overall responses (complete response+partial response) versus 75%, respectively (P < 0.001). At the time of an analysis performed in July 1995 (after a median follow-up period of 37 months), overall survival was also longer in the HD-CLB treatment arm (median survival, 68 months) than in the CHOP treatment arm (median survival, 47 months) (P < 0.005). Toxicity was acceptable and comparable in the two treatment arms. CONCLUSIONS: The current study showed that HD-CLB is an effective and well-tolerated therapeutic option for patients with advanced and/or progressive CLL. Therefore, the authors recommend its wider use, possibly in comparison with and/ or in combination with new therapeutic agents, such as purine analogues.