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Despite the high efficiency of tyrosine kinase inhibitors (TKI), some patients with chronic myeloid leukemia (CML) will display residual disease that can become resistant to treatment, indicating intraclonal heterogeneity in chronic-phase CML (CP-CML). To determine the basis of this heterogene-ity, we conducted the first exhaustive characterization of the DNA methyla-tion pattern of sorted CP-CML CD34 + CD15 A (immature) and CD34 A CD15 + (mature) cells at diagnosis (prior to any treatment) and compared it to that of CD34 + CD15 A and CD34 A CD15 + cells isolated from healthy donors (HD). In both cell types, we identified several hundreds of differentially methylated regions (DMRs) showing DNA methylation changes between CP-CML and HD samples, with only a subset of them in common between CD34 + CD15 A and CD34 A CD15 + cells. This suggested DNA methylation variability within the same CML clone. We also identified 70 genes that could be aberrantly repressed upon hypermethylation and 171 genes that could be aberrantly expressed upon hypomethylation of some of these DMRs in CP-CML cells, among which 18 and 81, respectively, were in CP-CML CD34 + CD15 A cells only. We then validated the DNA methylation and expression defects of selected candidate genes. Specifically, we identified GAS2, a candidate oncogene, as a new example of gene the hypomethylation of which is associated with robust overexpression in CP-CML cells. Altogether, we demonstrated that DNA methylation abnormalities exist at early stages of CML and can affect the transcriptional landscape

4Service d'Hematologie, Hopital Edouard Herriot, Lyon, France Abstract: Prior studies have shown that multidrug resistance gene products may be detected in acute myeloid leukemia (AML) cells, and are associated with poor response to therapy. We studied whether P-170 expression was associated with in vitro daunorubicin (DNR) accumulation and sensitivity of leukemic clonogenic cells (CFU-L) to DNR in 16 newly diagnosed AML samples. P-170 expression was assessed by indirect immunofluorescence using the monoclonal antibody MRK16. DNR cellular content was measured by flow cytometry after short incubation with increasing concentrations of DNR, and was not correlated with P-170 expression, although there was a trend for higher values in P-170-negative samples. The sensitivity of CFU-L was studied in a semisolid culture assay by calculating the dose of DNR inhibiting the growth of 90% of CFU-L (D90). The D90 was significantly higher in P-170-positive than in P-170-negative samples (mean = 1.68 ± 0.42 pg/ml versus 0.97 ± 0.35 μg/ml respectively, p< 0.005). Eight of 9 cases achieving complete remission (CR) after intensive chemotherapy were P-170-negative, whereas 7 of 7 nonresponders were P-170-positive (p< 10−5). D90 was significantly lower for patients achieving CR than for those who did not achieve CR (1.12 ± 0.55 μg/ml versus 1.59 ± 0.37 μg/ml, p = 0.04). It is concluded that P-170 expression is correlated with in virto resistance of clonogenic cells to DNR and may be one mechanism of resistance to chemotherapy.

Summary: A case of disseminated Scedosporium apiospermum infection in a patient undergoing bone marrow transplantation for chronic myeloid leukemia is reported. Because of graft rejection, the patient received a second transplantation 35 days after the first one, at which time he had a high fever and abdominal pains. A pulmonary infiltrate developed seven days later and S. apiospermum was grown from a bronchoalveolar lavage. The patient died 25 days after the second transplant with diffuse pulmonary infiltrates, renal failure and neurologic symptoms. An autopsy revealed a disseminated S. apiospermum infection, involving the lungs, kidneys, liver, spleen, gastrointestinal tract and the brain. The pathogen was resistant to amphotericin B and 5-fluorocytosine, but susceptible to miconazole and ketoconazole. Zusammenfassung Es wird uber einen Fall von disseminierter Scedosporium apiospermum-Infektion in einem Patienten berichtet, der wegen einer chronischen myeloischen Leukamie eine Knochenmarktransplantation erhalten hatte. Wegen der Transplantatabstosung erhielt der Patient 35 Tage nach der ersten eine zweite Transplantation; zu dieser Zeit hatte er hohes Fieber und Unterleibsschmerzen. Sieben Tage spater entwickelt sich ein Lungeninfiltrat, und S. apiospermum wurde aus einer Bronchiallavage gezuchtet. 25 Tage nach der zweiten Transplantation verstarb der Patient mit diffusen Lungeninfiltraten, Nierenversagen und neurologischen Symptomen. In der Autopsie fand sich eine disseminierte S. apiospermum-Infektion in der Lunge, den Nieren, der Leber, der Milz, dem Gastrointestinaltrakt und im Gehirn. Der Erreger war resistent gegenuber Amphotericin B und 5-Fluorcytosin, aber empfindlich fur Miconazol und Ketoconazol.

Summary: A case of invasive Geotrichum capitatum infection is reported; a young patient had an acute leukemia for which he received a chemotherapy, and presented sepsis with blood cultures for Geotrichum capitatum, namely Dipodascus spicife; this pathogen only described in cactus rot, is responsable for the first case of a human disseminated infection reported in literature. Then he developed a splenic and epididymic infection, with positive cultures for Geotrichum capitaturn after splenectomy and castration. Treatment with amphotericin B and itraconazole was started with low minimal inhibitory concentration (0.1 μg/ml). The patient died of massive hemoptisis. Autopsy findings demonstrated a lung, brain and kidneys seeding. Zusammenfassung: Es wird uber eine invasive Geotrichum capitatum-Infektion berichtet. Ein Patient mit akuter myeloischer Leukamie, gegen die er eine Che-motherapie erhalten hatte, entwickelte eine Sepsis, bei der Geotrichum capitatum (Dipodascus spicifer) in Blutkulturen nach-gewiesen werden konnte; dieser Pilz, bisher als Erreger der Kaktusfaule beschrieben, erwies sich als die Ursache der ersten am Menschen beschriebenen disseminierten Infektion. Der Patient entwickelte eine Milz- und Nebenhodeninfektion mit kultu-rellem Nachweis nach Splenektomie und Kastration. Es wurde eine Behandlung mit Amphotericin B und Itraconazol bei niedri-gen MHK-Werten (0, l μg/ml) eingeleitet. Der Patient verstarb unter massiver Hamoptoe. Autoptisch wurde Pilzbefall auch der Lunge, des Gehirns und der Nie-ren nachgewiesen.

Excessive apoptosis is implicated in the pathogenesis of myelodysplastic syndromes (MDS). We assessed by flow cytometry the expression of several members of the Bcl-2 family in bone marrow mononuclear cells (BMMNC) of 168 MDS samples at diagnosis. The proteins studied were Bcl-2, Bcl-xL (anti-apoptotic), Bax, Bad, Bak, and Bcl-xS (pro-apoptotic). The percentage of BMMNC expressing Bcl-2 and Bcl-xL was higher in refractory anemia with excess of blasts (RAEB), RAEB in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML) than in refractory anemia (RA) and RA with ringed sideroblasts (RAS). Conversely pro-apoptotic proteins Bad, Bak, and Bcl-xS were detected in a higher percentage of cells in RA and RAS. RA and RAS were associated with an increased Bcl-xS/Bcl-xL ratio. The expression of anti-apoptotic proteins was also correlated with that of CD34 and P170 and with the percentage of blast cells. Two-color analyses demonstrated that CD34 and Bcl-2 were usually expressed in the same cells. No significant correlation was found with cytogenetic abnormalities. Higher expression of pro-apoptotic Bcl-2-family proteins (Bak, Bad, Bcl-xS) and higher Bcl-xS/Bcl-xL ratio were associated with longer survival and decreased risk of leukemic transformation in univariate analysis, whereas expression of anti-apoptotic proteins was associated with decreased survival. Consequently Bcl-2 proteins expression was well correlated with the International Prognostic Scoring System (IPSS). Our data confirm that the control of apoptosis is deregulated in MDS cells. Moreover, the study of markers such as CD34 (or Bcl-2), Bcl-xL, and Bcl-xS provides additional prognostic information.

We report three cases of acute leukaemia with t(4;11) (q21;q23), one of them of undifferentiated and the other two of lymphoid phenotype, occurring after adjuvant radiochemotherapy for breast cancer (two cases) or occupational exposure to radiation (one case). Although the myeloid phenotype and characteristic chromosomal anomalies usually observed in secondary leukaemia were lacking, our observations raise the possibility of causal relationship between exposure to carcinogens and the occurrence of a leukaemia with t(4;11).

In an attempt to rationalize the use of therapy in acute myeloblastic leukemia (AML) in elderly patients, 69 cases of primary AML in patients older than 60 years of age were reviewed retrospectively. Therapy was empirical and 12 patients received supportive care (SC) only, 35 received aggressive chemotherapy (AC), and 22 received low-dose cytosine arabinoside (LD-araC). Patients receiving SC only often had a poor Karnofski index and their median survival was 17 days. Aggressive chemotherapy yielded complete remissions (CR) in 48% of the patients, whereas 23% of the patients had resistant disease (RD) and 29% had other failures (OF). Low-dose araC, which was administered to patients significantly older than those receiving AC, yielded 23% CR, 68% RD, and 9% OF, with important hematologic toxicity in most patients. Median survival was 211 days in patients receiving AC and 235 days in patients treated with LD-araC. Survival beyond 2 years from diagnosis was noted in the AC group only. A low Karnofski index was the strongest factor in poor prognosis, while age was not a prognostic factor. The initial characteristics of the patients did not allow us to define groups of patients who should be treated by either AC or LD-araC. We concluded that the decision to treat patients actively should rely more on the patient's general condition and socio-economical criteria than on age.

The clinical significance of surface markers was investigated in 145 cases of acute myeloid (AML) or undifferentiated leukaemia (AUL), using a panel of six monoclonal antibodies directed to NHL-30.5 antigen (expressed on poorly differentiated myeloid cells), CD13, CD14, CD15, CD33 and CD34 antigens. Expression of CD14 was correlated with higher leucocyte count, higher serum lactate dehydrogenase level and presentation with extramedullary disease. There was no strict correlation with the French-American-British classification. However, the expression of CD14 was associated with monocytic subtypes. CD15 was mainly expressed in M2 and M3 subtypes, and NHL-30.5 and CD34 antigens in AUL and M1 leukaemias. All patients were treated with the same intensive induction treatment. Staining by three antibodies had a prognostic value. The complete remission (CR) rates were 38% (26/68) in NHL-30.5-positive versus 75% (62/77) in NHL-30.5-negative cases (P less than 10(-5), 50% (37/74) in CD34-positive versus 72% (51/71) in CD34-negative cases (P = 0.007) and 70% (77/110) in CD15-positive versus 31% (11/35) in CD15-negative cases (P less than 10(-4). Expression of NHL-30.5 and CD34 antigen was associated with shorter survival (P less than 10(-3) and P less than 10(-2) respectively), whereas survival was longer in CD15-positive cases (P less than 10(-3). In multivariate analysis, expression of NHL-30.5 antigen, absence of CD15, and high LDH level were associated with poor survival. CR duration was not influenced by any of the factors studied, including antigen expression. These results suggest that leukaemias with less differentiated phenotype have a lower response rate to induction treatment.

A 26-year-old man with hypereosinophilic syndrome who had initial neurologic, cardiac, and pulmonary dysfunction, high eosinophil count, thrombocytopenia, and bone marrow fibrosis had only a transient response to conventional treatment with corticosteroids and hydroxyurea. He therefore received human lymphocyte antigen-identical allogeneic bone marrow transplantation (BMT) after conditioning with cytoxan and fractionated total body irradiation. Hematologic recovery was prompt, with normalization of blood counts and bone marrow. The patient died less than 3 months after transplantation from diffuse cytomegalovirus infection. Potential interest of BMT in patients with resistant hypereosinophilic syndrome and features of poor prognosis is discussed.