Angelo Guerrasio

Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients.

In order to verify if quantitative assessment of the WT1 transcript amount by the real time quantitative PCR (RQ-PCR) can be used as a marker for minimal residual disease detection, the WT1 transcript amount was determined in BM and PB samples of patients with myeloid and lymphoid acute leukemia, in normal controls, in regenerating bone marrow samples and in purified CD34-positive cells from normal subjects. In 10 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment, we performed a simultaneous analysis of the WT1 and of the fusion-gene transcript at sequential time intervals during follow-up. Sequential WT1 analysis was also performed in five AML patients lacking additional molecular markers. The data obtained show that normal and regenerating BM samples and purified CD34-positive cells consistently express minimal amounts of WT1 transcript and that this is extremely low and frequently undetectable in normal PB. By contrast, high levels of WT1 expression are present in the BM and PB samples of all acute leukemia (AL) cases at diagnosis. The WT1 levels during follow-up were found to follow the pattern of the other molecular markers (fusion gene transcripts) used for MRD monitoring and increased WT1expression in the BM and/or PB during follow-up of AL patients was always found to be predictive of an impending hematological relapse.

Assessment of minimal residual disease (MRD) in CBFbeta/MYH11 -positive acute myeloid leukemias by qualitative and quantitative RT-PCR amplification of fusion transcripts

The inv(16)(p13q22) chromosomal rearrangement associated with FAB M4Eo acute myeloid leukemia (AML) subtype is characterized by the presence of the CBFbeta/MYH11 fusion transcript that can be used to detect minimal residual disease (MRD). However, qualitative RT-PCR studies of MRD have so far produced conflicting results and seem of limited prognostic value. We have evaluated retrospectively MRD in a large series of CBFbeta/MYH11-positive patients employing both qualitative and quantitative (real-time PCR) approaches. 186 bone marrow samples from 36 patients were examined with a median follow-up of 27.5 months; 15 patients relapsed during follow-up. In qualitative studies, carried out by ‘nested’ RT-PCR assay, all patients in complete remission (CR) immediately after induction/consolidation therapy were found to be PCR positive. However, follow-up samples at later time points were persistently negative (except one case) in patients remaining in continuous CR (CCR) for more than 12 months. 16 patients were evaluated by quantitative real-time PCR assay: CBFbeta/MYH11 transcript copy number was normalized for expression of the housekeeping gene ABL, expressed as fusion gene copy number per 104 copies of ABL. A 2–3 log decline in leukemic transcript copy number was observed after induction/consolidation therapy. After achieving CR, the mean copy number was significantly higher in patients destined to relapse compared to patients remaining in CCR (151 vs 9, P < 0.0001 by Mann–Whitney test). Moreover, in CCR patients, the copy number dropped below the detection threshold after the treatment protocol was completed and remained undetectable in subsequent MRD analysis in accordance with results obtained by qualitative RT-PCR. On the contrary, in the seven patients who relapsed, the copy number in CR never declined below the detection threshold; thus a cut-off value discriminating these two groups of patients could be established. The findings of our study, if confirmed, might confer an important predictive value to quantitative real-time PCR determinations of MRD in patients with inv(16) leukemia.

The common TEL/AML1 rearrangement does not represent a frequent event in acute lymphoblastic leukaemia occurring in children with Down syndrome

Individuals with constitutional trisomy 21 (Down syndrome) are at increased risk of developing acute leukaemias, both of myeloid and lymphoid lineage. Although the cause of leukaemia in Down syndrome (DS) remains unknown, potential candidate genes include the ones on chromosome 21, and in particular AML1, the rearrangement of which in the t(8,21) is associated with the French–American–British (FAB) classification M2 subtype of acute myeloid leukaemia (AML) in the general population and has been described in Down patients with AML-M2. Recently, a new rearrangement involving AML1, the t(12;21), producing the TEL/AML1 hybrid transcript, has been described by molecular analysis as the most recurrent genetic lesion in childhood acute lymphoblastic leukemia (ALL). In order to investigate whether the t(12;21) could give a molecular clue as to the precise basis of the etiologic association between DS and acute lymphoblastic leukemia, we tested a series of 11 consecutive cases of ALL in DS children for the presence of the TEL/AML1 transcript, by RT-PCR analysis. We report absence of the TEL/AML1 rearrangement among the 11 cases tested. This data may be suggestive of alternative pathways involved in the pathogenesis of ALL in children with constitutional trisomy 21.

Genetic analysis of p53 and RB1 tumor-suppressor genes in blast crisis of chronic myeloid leukemia.

SummaryWe have investigated the involvement of the p53 and RB1 tumor-suppressor genes in 26 cases of chronic myeloid leukemia (CML) blast crisis, including 17 myeloid, eight lymphoid, and one megakaryoblastic crisis. The presence of p53 mutations in exons 5 through 9 was tested by the PCR-single-strand conformation polymorphism (SSCP) assay, followed by PCR-direct sequencing; in addition, loss of heterozygosity (LOH) at 17p13, the site of the p53 gene, was assayed by Southern blot. Given the variability of the mechanisms of inactivation of the RB1 gene in human tumors, a combination of Southern blot and mutational analysis by PCR-SSCP was used. p53 mutations were restricted to one case of myeloid blast crisis, showing a CGC→TGC (Arg→Cys) mutation at codon 283; two additional cases displayed LOH at 17p13 in the absence of p53 mutations. No molecular lesions of the RB1 gene were detected in any of the cases analyzed. These data indicate that inactivation of p53 and RB1 is a rare event in the molecular pathogenesis of CML acute transformation.

HHV8-positive, HIV-negative multicentric Castleman’s disease: early and sustained complete remission with rituximab therapy without reactivation of Kaposi sarcoma

Multicentric Castleman’s disease (MCD) is a rare lymphoproliferative disorder with systemic symptoms and poor prognosis and is characterized by an abnormal proliferation of polyclonal plasmablasts in the mantle zone of B-cell follicles. The disease is found primarily in chronic HIV carriers and is usually strictly associated with human herpes virus type 8 (HHV-8) coinfection, which is believed to play a key role in the pathogenesis of MCD. The disease is also diagnosed in HIV-negative patients, who are usually elderly or immunosuppressed; however, in about half of these cases, no evidence of HHV8 infection is found. The anti-CD20 monoclonal antibody rituximab is now the preferred treatment for HIV-positive MCD. However, it is not clear whether rituximab is effective in HIV-negative patients with MCD, particularly in the HHV8-positive subset. We report here the clinical and biologic courses of two HIV-negative, HHV8-positive patients with MCD who were treated with rituximab. In both cases, a significant clinical improvement was observed after the first two infusions, which was shortly followed by a drop in HHV8 viremia to undetectable levels. Both patients underwent complete clinical remission, which persisted without relapse at 30 and 9 months of follow-up, respectively. No reactivation of the Kaposi sarcoma found in a lymph node of one of the patients was observed. Our report, along with additional data present in the literature, suggests that rituximab may be an appropriate and safe first-line therapy for HIV-negative, HHV8-positive MCD.

Molecular Defects Associated with the Acute Phase CML

Parts of the Bcr/Abl hybrid transcript supposed to be important for its transforming ability were sequenced in a series of CML blast crises, in order to evaluate the possible presence of alterations responsible for the disease transition from the chronic to the acute phase. In addition, the N- and Ki-ras as well as the p53 involvement was investigated by exploring their structure and expression in the same patients. We used traditional types of molecular analysis including Southern and Northern blot, together with methods that allow a rapid detection of point mutations and microdeletions, such as SSCP, single strand conformation polymorphism and direct sequencing. The results obtained may be summarized as follows: no alterations were found in the parts of the Bcr/Abl transcripts investigated in the present study (SH2, SH3 and the region surrounding codon 832); p53 alterations were observed in 5% and N- and Ki-RAS mutations in 5% of the cases examined. These molecular defects are therefore responsible for the clinical progression of the Ph1-positive CML only in a minority of cases.

Polyclonal haemopoieses associated with long-term persistence of the AML1-ETO transcript in patients with FAB M2 acute myeloid leukaemia in continous clinical remission.

Summary. The t(8;21) (q22;q22) translocation is a recurring chromosomal abnormality observed in about 20‐40% of AML patients with subtype FAB M2 (AML‐M2). The molecular facet of this translocation is represented by the formation of a new hybrid gene, the AML1‐ETO, which is regularly transcribed in a chimaeric mRNA and translated into a new fusion protein believed to have a key role in the pathogenesis of this type of leukaemia. We looked for the presence of AML1‐ETO transcripts, by RT‐PCR, in 49 unselected patients affected by AML‐M2 diagnosed at various Italian Institutions. A hybrid transcript was detected in 11 cases (23%). Minimal residual disease status was investigated in three patients in continuous complete remission (CCR) after a median follow‐up of 44 months; at least one sample from each subject was found positive for the AML1‐ETO transcript suggesting a long‐term persistence of t(8;21) leukaemic cells.

PH Positive Acute Lymphoblastic Leukemia in Adults: Molecular and Clinical Studies

Fifty-six patients with ALL were investigated for bcr involvement by PCR. Breakpoints were found in 15 patients (26.8%). There were no differences in clinical and hematologic features or the percentages of complete response (CR) between the Ph+ and Ph- cases. The duration of CR was 6 and 8 months, respectively. In 7/9 Ph1 relapsed ALL we observed increased expression of myeloid markers and 2/9 showed a switch of cytotype (Ly-->My). In none of the 13 Ph- relapsed ALL patients did we observe these findings. 7/15 of Ph+ cases expressed P190 and mRNA ela2 and 8/15 patients showed P210, with mRNA b3a2 in 5 and b2a2 in 3, respectively. The percentage of CR was 57% in the P190+ and 87% in the P210+ group. Investigation of more Ph1+ ALL cases treated with a uniform protocol should be performed in the future in order to determine whether any such biological and clinical differences exist.

CHRONIC NEUTROPHILIC LEUKAEMIA ASSOCIATED WITH POLYCYTHEMIA VERA: PATHOGENETIC IMPLICATIONS AND THERAPEUTIC APPROACH

therapy is related to her last two remissions. It is interesting to REFERENCES note that although her serum ferritin levels at her presentation in October 1988 and now in remission are similar, the transferrin saturation is quite different (76% and 30% respectively). A possible hypothesis is that the iron-loaded mitochondria in the developing erythroblasts are responsible for malfunction and cell death and that remission has occurred when desferrioxamine treatment has reduced iron supply to the bone marrow. It could even be postulated that a vicious circle occurs whereby that as the haemoglobin falls, macrophage iron stores would increase, and the transferrin saturation would also tend to increase with erythroid hypoplasia. The iron data in relationship to her future course may help resolve the relationship between her iron status and sideroblastic anaemia. However, the primary cause of her sideroblastic anaemia remains unknown, whatever the relationship of her disease to desferrioxamine treatment. We would be interested to learn of other cases of relapsing sideroblastic anaemia and whether they have any features in common with this case. Hines, J.D. (1976) Effect of pyridoxine plus chronic phlebotomy on the function and morphology of bone marrow and liver in pyridoxine-responsive sideroblastic anaemia. Seminars in Haemato-

BCR/ABL Transcripts and Leukemia Phenotype: An Unsolved Puzzle

The Philadelphia chromosome, arising as a consequence of the t(9;22) translocation, is one of the most frequent and certainly the most known cytogenetic abnormality present in human hematological malignancies. Unlike the vast majority of the other translocations, its presence is not restricted to a specific leukemia phenotype, but is found associated with chronic myelogenous leukemia as well as with a large percentage of acute lymphoblastic leukemias, particularly in elderly patients. Although its molecular counterpart is always represented by a rearrangement between the BCR and the ABL genes, this shows a certain degree of molecular variability. The pathogenetic relationship with the different leukemia phenotypes which have been found to be associated still awaits to be fully elucidated. However, a number of old and more recent observations seem to suggest that not only qualitative differences in the type of BCR/ABL proteins expressed, but also quantitative variations in their total level within the cells may have an important role in determining the leukemia phenotype.