Françoise Valensi

Splenic lymphoma with villous lymphocytes: clinical presentation, biology and prognostic factors in a series of 100 patients

The diagnosis of splenic lymphoma with villous lymphocytes (SLVL) was assessed by a panel of cytologists in a series of 100 patients. Clinical and biological characteristics were analysed in relation to prognosis.

Sequential chemotherapy by CHOP and DHAP regimens followed by high-dose therapy with stem cell transplantation induces a high rate of complete response and improves event-free survival in mantle cell lymphoma: a prospective study.

Mantle cell lymphoma (MCL) is a distinct clinico-pathological entity with a poor prognosis. We have conducted a prospective study in patients with MCL to evaluate a therapeutic strategy in which CHOP polychemotherapy was followed by DHAP if CHOP failed to induce complete remission. Responding patients then proceeded to an intensification therapy with autologous peripheral blood stem cell transplantation (APBSCT). Twenty-eight consecutive patients with newly diagnosed aggressive MCL were included. After four cycles of CHOP regimen, two complete responses (CR) were obtained (7%) and 14 (50%), five (18%) and seven (25%) patients achieved partial (PR), minor (MR) and no response, respectively (one patient died from septic complications during CHOP induction). The two patients in CR after CHOP underwent intensification with TBI, high-dose cyclophosphamide–etoposide and APBSCT. The other twenty-five patients received DHAP and in this group a response rate of 92% (21 CR (84%), two PR (8%)) was observed. Two patients had progressive disease. The twenty-three responding patients received high-dose therapy (TAM8 regimen: TBI–cytarabine–melphalan) followed by APBSCT. One of the two partial responding patients achieved CR after TAM8. After a median follow-up of 47.6 months (range, 14–70), seven patients have relapsed. Our data confirm that: (1) CHOP regimen induces a low CR rate in MCL; (2) CHOP plus DHAP appears to be much more efficient and allows a large proportion of patients to proceed to high-dose therapy in CR; (3) consolidation therapy including TBI and high-dose Arac-C followed by APBSCT may improve event-free survival.

Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors

Erythropoiesis is positively regulated by stem cell factor, interleukin 3, and erythropoietin, which synergize to allow the production of hemoglobinized red blood cells from erythroid progenitors. In contrast, interferon gamma, tumor necrosis factor alpha, and transforming growth factor B(1), (TGF-beta(1)) are powerful inhibitors of erythropoiesis. Interferon gamma and alpha act principally by inducing apoptosis. The aim of this study was to elucidate the mechanisms by which TGF-beta(1) inhibits erythropoiesis. We used an in vitro serum-free system of human red blood cell production. From a virtually pure population of CD36(+) erythroid progenitors, stem cell factor, interleukin 3, and erythropoietin allowed massive proliferation (x300) and promoted terminal red blood cell differentiation. We show here that TGF-beta(1) (2 ng/mL) inhibited the growth of CD36(+) cells by 15-fold. TGF-beta(1) markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression. Furthermore, May-Grünwald-Giemsa staining and ultrastructural analysis revealed that TGF-beta(1) induced full differentiation toward normal enucleated red cells even in the absence of macrophages. This acceleration of erythroid differentiation did not modify the pattern of hemoglobin chains expression from adult or fetal erythroid progenitors. Analysis of apoptosis, cell cycle and Ki-67 expression showed that TGF-beta(1) inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle. We showed that TGF-beta(1) is a paradoxical inhibitor of erythropoiesis that acts by blocking proliferation and accelerating differentiation of erythroid progenitors.

Acute monocytic leukemia chromosome studies

Cytogenetic studies have been performed on 34 acute monocytic leukemia (M5) patients, 24 of the a type and 10 of the b type. No chromosomal abnormalities were found in 12 cases, in spite of the fact that the mitoses concerned monocytes. Different chromosomal aberrations were present in the other cases. In 12 of them, an abnormality of the chromosome 11 long arm was observed (mainly in the poorly differentiated type of M5), on bands q22-q24 in nine cases and on band q14 in three cases. The chromosome 11 long arm thus appears preferentially rearranged in M5 although this is not apparent in every case. Concomitant study of the mitoses with cytological and cytogenetic techniques suggests that erythroblasts may not be involved in the M5 leukemic process.

Molecular detection of t(8;21)/AML1-ETO in AML M1/M2: correlation with cytogenetics, morphology and immunophenotype

The t(8;21) identifies a subgroup of acute myeloid leukaemia (AML) with a relatively good prognosis which may merit different treatment. It is associated predominantly, but not exclusively, with AML M2, and corresponds to rearrangements involving the AML1 and ETO genes. AML1‐ETO positive, t(8;21) negative cases are well recognized but their incidence is unknown. In order to determine optimal prospective AML1‐ETO RT‐PCR screening strategies, we analysed 64 unselected AML M1 and M2 cases and correlated the results with other biological parameters. Molecular screening increased the overall detection rate from 8% to 14%. AML1‐ETO was found in 3% (1/32) of AML M1 and 25% (8/32) of M2, including three patients without a classic t(8;21) but with chromosome 8 abnormalities. It was more common in younger patients. Correlation with morphology enabled development of a scoring system which detected all nine AML1‐ETO‐positive cases with a false positive rate of 7% (4/55). Although certain AML1‐ETO‐positive cases demonstrated characteristic immunological features (CD19 and CD34 expression, CD33 negativity), each of these markers was insufficiently specific to permit prediction in an individual case. We conclude that initial routine prospective molecular screening for AML1‐ETO in all AMLs, combined with standardized morphological and immunological analysis, is desirable in order to produce improved prognostic stratification and to determine whether screening can ultimately be restricted to appropriate subgroups.

Human herpes virus 8 (Kaposi's sarcoma herpes virus) and malignant lymphoproliferations in France: a molecular study of 250 cases including two AIDS-associated body cavity based lymphomas.

The new human herpes virus 8 (HHV8) was recently detected in cases of body cavity based lymphoma (BCBL), a rare B cell lymphoma, mostly AIDS-associated. We investigated for HHV8 DNA sequences a series of 250 B or T cell lymphoproliferative malignancies, as seen in France, including 126 leukemias and 124 lymphomas (232 non-AIDS-associated and 18 AIDS-associated tumors). HHV8 sequences were detected in only three patients. The first two were homosexual males, HIV-infected since 1985 who suffered from a BCBL initially characterized in one case by a pleural lymphomatous effusion and a peritoneal one in the other case. A high level of HHV8 copies was detected in the tumoral cells of these two BCBL. In contrast, in the third positive patient who had an AIDS-associated immunoblastic lymphoma, the HHV8 sequences level was quite low. In the two BCBL patients, the HHV8-infected clonal B cells had a large immunoblastic feature with an indeterminate phenotype and were also infected by Epstein–Barr virus. In one BCBL case, a semiquantitative PCR analysis revealed that the HHV8 sequences were much more abundant in the effusion tumor cells than in the cutaneous Kaposi’s biopsy while no HHV8 sequence was detectable in the peripheral blood lymphocytes. This study reports HHV8-associated BCBL in European AIDS patients and confirms that HHV8 is present at a high copy number in the tumoral B cells of this malignancy. Furthermore, HHV8 does not seem to play a pathogenic role in any of the other T or B malignant lymphoid neoplasias studied so far. This study also stresses the necessity for quantification studies in interpretation of a positive PCR analysis for HHV8 sequences, especially in patients at risk for HIV infection or Kaposi’s sarcoma.

Rapid, multifluorescent TCRG Vγ and Jγ typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations

Detection of clonal T cell receptor γ (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10−3 at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V–J rearrangements from childhood cases. We describe rapid, multifluorescent Vγ and Jγ PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of Vγ and Jγ segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10−2–10−3 level. We demonstrate preferential V–J combinations but no difference in V–J usage between children and adults, nor between SIL-TALI-negative and -positive cases. A combination of fluorescent multiplex and Vγ–Jγ-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.

Absence or low expression of fas-associated protein with death domain in acute myeloid leukemia cells predicts resistance to chemotherapy and poor outcome

In acute myeloid leukemia (AML), coexpression of death receptors and ligands of the tumor necrosis factor (TNF) receptor/TNF-α superfamily on leukemic cells after chemotherapy is not always accompanied by apoptosis, suggesting that the apoptotic death receptor signaling pathway is disrupted. Because Fas-associated protein with death domain (FADD) is the main adaptor for transmitting the Fas, TNF-related apoptosis-inducing ligand receptors, and TNF receptor 1 death signal, expression of FADD was analyzed by Western blot and immunocytochemistry in leukemic cells of 70 de novo AML patients treated with the European Organization of Research and Treatment of Cancer AML-10 randomized trial before initiation of induction chemotherapy. Thirty seven percent of patients (17 of 46) with FADD negative/low (FADD−/low) leukemic cells had a primary refractory disease compared with 12% of FADD+ patients (3 of 24; P = 0.05). FADD−/low expression was significantly associated with a worse event-free survival [EFS (P = 0.04)] and overall survival (P = 0.04). In multivariate analysis, FADD−/low protein expression was independently associated with a poor EFS and overall survival (P = 0.002 and P = 0.026, respectively). Importantly, FADD−/low protein expression predicted poor EFS even in patients with standard- or good-risk AML (P = 0.009). Thus, we identified low or absent expression of the FADD protein in leukemic cells at diagnosis as a poor independent prognostic factor that can predict worse clinical outcome even for patients with standard- or good-risk AML.

Recurrence of OTT-MAL fusion in t(1;22) of infant AML-M7.

Translocation t(1;22)(p13;q13) is associated with a peculiar subtype of acute megakaryocytic leukemia (M7) occurring in infants. We have recently characterized a fusion gene, OTT–MAL, resulting from this translocation. We now report three additional cases and show that this gene fusion is present in all five t(1;22) cases studied to date. Nucleotide sequence analysis of two translocation breakpoints suggests a nonhomologous end joining mechanism in the genesis of this translocation and reveals a noncanonical topoisomerase II‐like consensus sequence within the OTT gene. FISH and PCR techniques described in this work are useful for identifying t(1;22) associated with M7.

The majority of myeloid‐antigen‐positive (My+) childhood B‐cell precursor acute lymphoblastic leukaemias express TEL‐AML1 fusion transcripts

The t(12;21) translocation fuses the TEL and AML1 genes and has been found in up to 28% of paediatric B‐cell precursor acute lymphoblastic leukaemias (BCP‐ALL). The AML1 gene is a transcription factor which regulates expression of several myeloid differentiation associated genes. A molecular analysis of TEL‐AML1, E2A‐PBX1, MLL‐AF4, BCR‐ABL expression and an immunophenotypic study of CD13/CD33 myeloid antigen expression have been performed prospectively on tumour cells from 96 paediatric BCP‐ALL patients. Percentages of CD13 or CD33 expressing leukaemic cells were found to be higher in TEL‐AML1 positive cases (n = 22) than in TEL‐AML1 negative (n = 74) cases (P < 0.001). In 22/96 cases (23%) >10% of neoplastic cells were found to express at least one of the two markers. In 14 of these cases (63%), TEL‐AML1 expression was detected, whereas t(4;11), t(11;19) and t(9;22) translocations were found by molecular methods in only three cases (14%). In four cases (18%) no molecular marker was found. These data show that TEL‐AML1 expression is significantly associated with myeloid antigen expression by leukaemic cells and suggests that the prognostic significance of myeloid antigen expression in paediatric ALLs should be re‐evaluated in the light of molecular cytogenetic markers.