Very early onset of an acute myeloid leukemia in an adult patient with B-cell lymphoblastic leukemia.

We report on a case of a 30-year-old male with acute B-lymphoblastic leukemia (B-ALL) with immunophenotype CD19(+), CD22(+), CD20(+), CD10(+), with aberrant expression of CD13 and CD117, and IgH gene rearrangements. Three months after treatment with GMALL-2003 and Ida/FLAG protocols bone marrow showed predominance of blasts with myeloid morphology and phenotype MPO(+), CD13(+), CD33(+), CD64(+), CD15(+), CD56(+), EVI-1 gene overexpression and lack of IgH rearrangements. The case is the first report of a very early emergence of myeloid leukemia during the induction treatment for B-ALL in an adult patient. Different pathogenetic mechanisms are discussed - clonal evolution or selection, lineage switch or development of a de novo or therapy-induced leukemia.

Secondary acute myeloid leukemia early after therapy for Hodgkin's disease--a case report.

A case of acute myeloid leukemia (AML) after successful therapy for Hodgkin's disease (HD) is reported. The patient was diagnosed with stage IIB HD at the age of 25. She was treated with chemotherapy and radiotherapy (RT), and complete remission (CR) was achieved. Seven months after the CR was obtained the patient developed AML. Knowing that the prognosis of patients with secondary AML (sAML) after primary HD is poor we decided to perform autologous peripheral stem cells' transplantation.

Induction of apoptosis by electrotransfer of positively charged proteins as Cytochrome C and Histone H1 into cells.

Cytochrome C (Cyt. C) is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown mechanism, but regulated by the bcl-2 family of proteins. The linker Histone H1 is another basic protein with highly conservative structure, composition, and equal molecular weight, not changed during the evolution. An attempt was made to understand better the apoptotic processes by electroloading of leukemic cells, such as K562, HL-60, and SKW3, and human lymphocytes with positively charged proteins, such as Cyt. C, Histone H1, and methylated BSA albumin (mBSA). The triggering apoptotic processes followed by MTT test, FACS analysis, and DNA fragmentation after the electrotransfer of these proteins into the cells were observed. Histone H1 and mBSA induce the release of Cyt. C from rat liver mitochondria. Cytochrome C release was higher when mitochondria were in "high-energy" state. It is supposed that release of Cyt. C from mitochondria is due to the mechanical rupture of the outer mitochondrial membrane, rich in negatively charged groups, predominately due to cardiolipin. The reason for the morphological rupture of the outer mitochondial membrane could be the rigidification and segregation of the membrane and the destroyed membrane asymmetries of both monolayers in the presence of positively charged proteins at higher linear charges such as Histone H1. We suggested that Histone H1, at a given moment of activated signaling for apoptosis, could be not transported to the nucleus and could lead to the release of Cyt. C from the mitochondria in the cytoplasm. It is temping to speculate that Histone H1 has other physiological extranuclear functions involved in apoptosis.

Acute megakaryoblastic leukaemia with extreme thrombocytosis and p190(bcr/abl)rearrangement.

Acute megakaryoblastic leukaemia (AML-M7) is an uncommon disease, composing 0.5-1.2% of newly diagnosed adult acute myeloid leukaemias (AML). It is characterised by higher incidence and complexity of cytogenetic abnormalities. We report a rare case of Philadelphia (Ph) chromosome-positive AML-M7, presenting with extreme thrombocytosis and having a poor outcome. The diagnosis was established on the basis of morphological and flow cytometry data for megakaryoblastic proliferation in the bone marrow. Cytogenetics revealed 47,XX,+8,t(9;22)(q34;q11), and p190(BCR-ABL)-rearrangement was detected. MDR1-gene overexpression was not demonstrated; however, the patient was resistant to therapy and died in 6 months. The reported case contributes to the overt heterogeneity of Ph-positive AMLs, which warrants further investigation and understanding.

Clonal heterogeneity in a patient with acute promyelocytic leukemia.

A 18-year-old man was diagnosed with acute promyelocytic leukemia (APL). The conventional cytogenetic analysis revealed normal karyotype 46, XY, t(15; 17). Reverse transcriptase polymerase chain reaction (RTPCR) identified PML-RARa chimeric transcripts. Complete remission (CR) was attained with 3 induction courses of Ara-C, daunorubicin and all-trans retinoic acid (ATRA). Three years later the patient relapsed. The blasts in bone marrow aspirate at relapse had AML-M3 morphology, and RT-PCR was positive for PML-RARa transcripts. The patient was treated with ATRA and daunorubicin without success. Two months later the blasts in bone marrow aspirate showed AML-M2 morphology, the karyotype was 47, XY, +8 and RT-PCR revealed the presence of AML1-ETO transcripts and absence of PML-RARa transcripts. The patient attained second CR with 3 induction courses -a course with Ara-C and daunorubicin and 2 courses with idarubicin, Ara-C and etoposide.

Application of reverse transcription polymerase chain reaction for analysis of multidrug resistance in patients with acute myeloblastic leukemia.

PURPOSE: To test the multidrug resistance 1 (MDR1) gene expression in acute myeloid leukemia (AML) patients using the reverse transcription polymerase chain reaction (RT-PCR) in order to assess the application of the method for routine screening. MATERIALS AND METHODS: 39 AML patients (mean age 43.9-/+18.2 years) and 10 healthy volunteers were studied by simultaneous amplification of the MDR1 and beta2-microglobulin (beta2-M) mRNA, as an internal control. Semi-quantitative characterization of MDR1 expression was done using a 4-grade scoring system: negative (-/+), weakly positive(-/+), moderately positive (+), strongly positive(++), according to the intensity of the MDR1 and beta2-M bands. RESULTS: The amplification of the MDR1 in healthy individuals yielded no products in 9 samples and in 1 case a (-/+) reaction was observed. In AML patients, RT-PCR revealed no MDR1 product (normal level) in 19 (48.7%) and (+)/(++) reaction (overexpression) in 15 (38.5%) patients. In the remaining 5 (12.8%) patients a (-/+) reaction was found, comparable to that of the (-/+) healthy individual (the level of MDR1 expression could not be defined). MDR1 overexpression was seen in 73.3% of the M1/M2 patients, and only in 14.3% of the M3 and M4/M5 patients. Besides, the MDR1(- )/(-/+)patients were significantly younger than MDR1(+)/ (++) patients (mean age 38.4-/+19.5/43.0-/+8.7 years, versus 51.1-/+16.7/65.0-/+4.3 years). CONCLUSION: RT-PCR allows for the identification of patients with moderate and higher levels of MDR1 overexpression, while the weak positive reactions require additional testing. Besides, our data support the observation that the level of expression might be associated with the French-American-British (FAB) subtype of AML and with the age of the patients.

Light microscopic detection of BCR-ABL transcripts after in-cell RT-PCR: fusion gene expression might correlate with clinical evolution of chronic myeloid leukemia.

A procedure for in-cell amplification of the hybrid BCR-ABL mRNA by reverse transcription and polymerase chain reaction (RT-PCR) without extraction of the nucleic acids was performed directly in fixed and permeabilized cells of leukemia patients (22 patients with Ph'-positive chronic myeloid leukaemia-CML and 1 with Ph'-positive acute leukaemia-AL, as well as 7 Ph'-negative cases) and Ph'-positive human leukaemia cell lines (K562, LAMA-84, BV173). The labelling of the amplified sequences was done employing biotinylated primers and a second PCR in a semi-nested fashion with a low number of cycles. An enzymatic system based on biotin-streptavidin-chromogen reaction was used for the detection of labeled PCR product, thus producing a coloured product, visible to the eye under a standard light microscope. All samples from patients with cytogenetic and molecular evidence of BCR-ABL rearrangement showed specific cytoplasmic staining at the site of the amplified hybrid transcripts. It allowed definite distinction between positive and negative cells. K562, LAMA-84, BV173 cells were characterized with strong diffuse staining while an interesting finding of the present study was the presence of variable quantities of colour product in patients' samples which might be due to different mRNA expression. Early and intermediate stages of myeloid maturation showed more intense reactivity. Cases with an aggressive course of accelerated or blast phase CML and AL were found to have a considerable subset of cells with strongly expressed signal while cases in chronic phase were characterised with uniform weak to moderate reaction. Our observations support the hypothesis that the amount of BCR-ABL transcript expression within neoplastic cells may play a role in dictating the eventual behaviour of the leukaemic clone. Future studies at a single cell level of larger series of consecutive cases with a follow up might be able to identify those patients who are prone to transformation and provide certain indications for further therapeutic decisions.

p16INK4A is regularly expressed in Hodgkin's disease: comparison with retinoblastoma, p53 and MDM2 protein status, and the presence of Epstein-Barr virus.

In order to understand better the possible role of cell-cycle regulating molecules in the pathogenesis of Hodgkin's disease (HD), the immunohistochemical distribution pattern of p16INK4A was investigated and compared with pRb, p53, and MDM2 protein status in 44 HD cases. Our findings were correlated to the presence of Epstein-Barr virus as detected by RNA in situ hybridization and clinicopathological parameters. p16INK4A protein immunoreactivity was found in all 44 cases with a proportion of Hodgkin-Reed-Sternberg (HRS) cells ranging from 30 to 90%. In 93% of the cases studied, pRb was detected in HRS, whereas all cases showed overexpression of p53. Almost all specimens (98%) were MDM2-positive as evaluated by 1B10 and/or IF2 monoclonal antibodies. EBER 1/2-transcripts were detected in 31.8% (14 of 44) of the examined samples. A significant correlation was observed between immunoreactivity of p16INK4A and MDM2 and the number of HRS cells (P = .0012 and P = .018, respectively). In a subgroup of cases, with p16INK4A expression in more than 50% of HRS cells, the percentage of pRb-positive neoplastic cells was inversely related to that of p16-positive ones (P = .007). No clinicopathological parameters or clinical prognostic indicators, including duration of response to therapy, were statistically related to the expression levels of any of the four proteins investigated or the presence of Epstein-Barr virus. Our findings suggest that p16 and pRb are regularly expressed and that their pathway in cell-cycle machinery seems to be intact in HD. However, further investigation is needed to shed light on the involvement of cell-cycle molecules in Hodgkin's lymphomagenesis and longer patient follow-up is required for conclusive prognostic correlation.

A new variant chromosomal translocation t(2;2)(p23;q23) in CD30+/Ki-1+ anaplastic large cell lymphoma.

A case with a complex chromosome abnormality with a t(2;2)(p23;q23) in CD30+/Ki-1+ anaplastic large cell lymphoma (ALCL) is described. This chromosome aberration has not been reported previously in neoplastic diseases and was associated with T-cell phenotype and involvement of the nasopharynx by the tumour.

Levels of expression of CAF7 (CD98) have prognostic significance in adult acute leukemia.

The levels of CD98 antigen expression were studied in 62 consecutive cases of adult acute leukemia including 24 acute lymphoblastic leukemia (ALL) and 38 acute myeloid leukemia (AML) using the monoclonal antibody CAF7 and flow cytometry. The mean follow-up was 13.5 months. The mean relative fluorescence intensity (MIF) of CAF7 varied between 6 and 83 channels (256 channels resolution). No correlation was established between CAF7 cell surface density and most of the predictive parameters such as age, sex, blood counts, immunophenotype, proliferative index (PI) or DNA index. Nevertheless expression of CAF7 correlated positively with survival duration (mean 210 vs 391 days, P = 0.048) and complete remission (CR) duration (mean 132 vs 361, days P = 0.032). The levels of CAF7 differed significantly between ALL and AML (P < 0.001), the ALL cases being all CAF7intermediate or CAF7high. In the AML group the low levels of CAF7 expression correlated with shorter CR duration (mean 132 vs 414 days, P = 0.017). The lack of correlation with other clinical and biological parameters suggested that CAF7 might have an independent prognostic significance in adult AML. Although PI was also positively related to survival duration (P = 0.02), it did not correlate with CR duration or the expression of CAF7. We suppose that the prognostic impact of CD98 is related to the control of cell growth and survival in which the molecule normally participates.

CD4+ acute undifferentiated leukaemia, probably early monoblastic type, developing in a patient with myelodysplastic syndrome and bone marrow fibrosis.

We report here a patient who presented with pancytopenia, hypercellular bone marrow and three-lineage dysplasia associated with an increase of reticulin fibres. After a 5-month period, anaemia and thrombocytopenia progressed very rapidly and the white blood count increased showing 45% blasts with monocytoid morphology, but cytochemically undifferentiated in nature. The immunophenotype revealed an unusual expression of CD4, CD36 and HLA-DR in the absence of any other myeloid or lymphoid lineage-associated markers. The patient died unexpectedly during the course of chemotherapy. The occurrence of CD4, CD36 and HLA-DR on the blast cells cannot determine the lineage of differentiation with certainty but provides some evidence that the leukaemic cells were probably derived from a very early monocytic progenitor with maturation arrest. These cells had apparently complex interactions with pathologic megakaryocytes and cytokine production.