Thierry Lavabre-Bertrand

Plasma proteasome level is a potential marker in patients with solid tumors and hemopoietic malignancies.

Proteasomes are nonlysosomal proteolytic structures that have been implicated in cell growth and differentiation. Abnormal expression levels of proteasomes have been described in tumor cells, and proteasomes can be detected and measured in plasma. The objective of this study was to characterize differences in proteasome levels between normal, healthy donors and patients with neoplastic disease and to correlate the findings with clinical status and other biologic markers of disease spread.

High plasma proteasome levels are detected in patients with metastatic malignant melanoma

Background  Proteasomes, nonlysosomal proteolytic structures, are implicated in cell growth and differentiation. An abnormal expression has been described in haematopoietic malignancies and in some solid tumours.

Diabetes insipidus revealing acute myelogenous leukaemia with a high platelet count, monosomy 7 and abnormalities of chromosome 3: a new entity?

Abstract: We describe three cases of acute myeloid leukaemia revealed by diabetes insipidus. The patients were 42, 38 and 39 yr old and they had marked hyperleukocytosis, circulating immature granular cells and a normal or elevated platelet count. The leukaemia was type AML‐M2 according to the FAB classification. Cytogenetic studies showed inversion of chromosome 3 (q21;q26) in 2 cases and a translocation (3;3)(q21;q29?) in the remaining case, both associated with monosomy 7. All the cerebral CT scans were normal. Complete remission was never achieved, and all three patients survived less than 14 months. Desmopressine therapy was active but treatment could not be reduced. The association of dysmegacaryopoiesis with a chromosome 3 abnormality and diabetes insipidus is probably not fortuitous and could represent a new entity.

Detection of membrane and soluble interleukin‐6 receptor in lymphoid malignancies

Summary. We studied the membrane expression of the gp80 chain of IL‐6 receptor (IL‐6R) by quantitative flow cytometry in chronic lymphocytic leukaemia (CLL) and leukaemic centrocytic lymphoma using a panel of seven monoclonal antibodies. IL‐6R was detected in 18/26 CLL cases and 4/7 lymphoma cases, with a mean antigen density <3000 molecules/cell. Multiple labelling experiments confirmed the IL‐6R expression by neoplastic cells. Specific mRNA was found by RT‐PCR in neoplastic cells. A specific ELISA test was designed using two anti‐IL‐6 receptor MAbs to measure the serum soluble IL‐6R (sIL‐6R) in CLL (n = 48), B‐cell non‐Hodgkins lymphoma (NHL; n = 40), and monoclonal gammopathy (MG; n = 32). SIL‐6R was higher in CLL (170±12.6ng/ml) in NHL (160 ± 12ng/ml) and MG patients (183±23ng/ml) than in age‐matched controls (100 ±5.6 ng/ml; P < 0.001) and higher in high‐grade than low‐grade NHL. No correlation was noted with a previous treatment. Among CLL cases the patients classified as stage B according to the Binets staging of the disease had the highest sIL‐6R values, thus suggesting a link with tumour cell mass.

Alpha-interferon therapy for congenital dyserythropoiesis type I

We report the case of a 28‐year‐old female followed for congenital dyserythropoiesis type I which required repeated transfusions. Alpha‐2a interferon treatment was started because of post‐transfusion chronic viral hepatitis type C. Following this treatment, haemoglobin level increased and reached normal value during the 24 weeks of interferon treatment. When interferon therapy was stopped, haemoglobin level returned to previous values, requiring more transfusions. Resumption of interferon therapy resulted again in a complete normalization of haemoglobin level. Erythrokinetic studies demonstrated a striking reduction of the ineffective erythropolesis, and electron microscopy study a reduction in nuclear structure abnormalities. To our knowledge, this is the first report of the efficacy of interferon in congenital dyserythropoiesis.

Long-term alpha interferon treatment is effective on anaemia and significantly reduces iron overload in congenital dyserythropoiesis type I

Abstract:  Interferon has been shown to be an effective treatment of congenital dyserythropoiesis type I (CDA‐I), but the optimal dose and the feasibility of this treatment remains to be determined. Here, in a 9‐yr follow‐up of a single patient, we show that interferon remains active during such a long period. The optimal dose of conventional alpha interferon could be evaluated at 2 million units twice a week. Pegylated interferon could be used as well at a dose of 30 μg/wk. During interferon treatment, serum and erythrocyte ferritin levels decreased progressively, and remained inversely correlated with haemoglobin levels. On repeated liver biopsies, iron overload could be normalized. Low dose interferon is a long‐term treatment of CDA‐I, and allows a significant decrease in iron overload, that could be interesting even in patients who are only moderately anaemic.

Spontaneous monoclonal immunoglobulin‐secreting peripheral blood mononuclear cells as a marker of disease severity in multiple myeloma

Peripheral blood from patients with multiple myeloma (MM) contains a small number of plasma cells related to the bone marrow tumour cells by their cytoplasmic immunoglobulin (Ig), their cell membrane antigen expression and/or their gene rearrangements, but hitherto the monoclonal Ig (M‐Ig) production by circulating cells has not been reported. Using a two‐colour ELISPOT assay, Ig‐secreting cells (Ig‐SCs) were detected in the blood of 28 MM and five Waldenstroms macroglobulinaemia (WM) patients. The number of cells that spontaneously produced an Ig isotype similar to that of the M‐Ig in serum was greater than that of the other Ig‐SCs. MM patients presented an excess of circulating heavy‐chain (α or γ) Ig‐SCs (0.38% of the PBMC) with κ or λ light chains (0.48%) compared with the number of cells secreting the other heavy‐ (0.02%) and light‐chain isotypes (0.03%). WM patients also presented high numbers of cells secreting the μ‐heavy‐chain isotype (0.66%). The Ig synthesized in vitro was characterized as monoclonal, and the M‐Ig secretory capacity of the peripheral blood cells was similar to that observed for Ig‐SCs from polyclonal activated B cells in vivo. The number of these monoclonal cells was significantly increased in patients in an advanced stage of MM (I/II vs. III, P < 0.001) and correlated with the serum beta‐2 microglobulin concentration (r = 0.69; P < 0.0003). The number of M‐Ig‐SCs in MM patients could be a useful marker for evaluating the progression of multiple myeloma.

Nested High-Resolution Melting Curve Analysis: A Highly Sensitive, Reliable, and Simple Method for Detection of Jak2 Exon 12 Mutations—Clinical Relevance in the Monitoring of Polycythemia

JAK2 exon 12 mutations are found in myeloproliferative disorders characterized by erythrocytosis. Lying in a 33-bp region and conserving the open reading frame, they often present a low allelic burden (<10%), which excludes screening with techniques such as allele-specific PCR or different sequencing protocols. High-resolution melting (HRM), a fast in-tube method, seems the most accurate routine technique for that. We describe a reliable and powerful nested HRM technique, independent of DNA preparation and with technical sensitivity of 100% (95% CI, 93% to 100%) and specificity of 96.7% (95% CI, 89.7% to 96.7%). Screening a cohort of 10 idiopathic erythrocytosis, 28 polycythemia vera, and 7 secondary erythrocytosis cases allowed the detection of 15 mutants, including 9 different mutations, of which 3 were unreported, all in the polycythemia vera group, and presented a characteristic profile: pure erythrocytosis associated with low serum erythropoietin. Threshold detection level ranged from 1% to 3% allelic burden, depending on the mutation. All of the HRM positive signals were found mutated by sequencing. Six of them (40%), however, required cloning before sequencing, because of low allelic burden. Classic techniques such as genomic sequencing may therefore miss patients with mutations. Given its sensitivity, HRM (and nested HRM) can be used in routine diagnosis and seems to be the most efficient of current techniques for detection of JAK2 exon 12 mutations.

On quantification of CD1a, HLA-DR, and HLA class I expression on viable human Langerhans cells and keratinocytes.

In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 +/- 19 x 10(3) molecules/cell) compared to viable Kc (785 +/- 110 x 10(3) molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 +/- 82 x 10(3) molecules/cell and 1600 +/- 133 x 10(3) molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases.

Exon 7 Deletion in the bcr-abl Gene Is Frequent in Chronic Myeloid Leukemia Patients and Is Not Correlated with Resistance against Imatinib

Chronic myeloid leukemia (CML) patients treated with imatinib develop frequent resistance generally due to a point mutation. Recently, large rearrangements of abl sequence have also been described. In this study, we focused on the complete deletion of exon 7. We screened for bcr-abldelexon7 in 63 resistant patients by high-resolution melting (HRM) analysis and direct sequencing. Moreover, we analyzed expression of abldelexon7 and bcr-abldelexon7 in 17 CML patients at diagnosis, 32 patients at resistance, and 20 negative controls by quantitative PCR or fragment length analysis. bcr-abldelexon7 was detected on 34 (54%) among 63 resistant patients by HRM, showing an increase in the sensitivity of screening, because only 3.2% could be detected by direct sequencing. This deletion was not associated with a point mutation (P = 0.3362). In addition, abldelexon7 was found in all tested samples with the same pattern of expression, suggesting an alternative splicing mechanism. In the bcr-abl component, there was no statistical difference between CML patients at diagnosis and resistant patients (P = 0.2815) as regarding bcr-abldelexon7 proportion, thus arguing against involvement of deletion in resistance. Moreover, among two patients harboring bcr-abldelexon7 at diagnosis, one experienced a complete disappearance of this transcript, and the other decreased >75% at resistance. In conclusion, bcr-abldelexon7 is frequently observed in CML patients when using sensitive techniques. It seems to be the result of an alternative splicing mechanism and to be independent from the occurrence of resistance. Mol Cancer Ther; 9(11); 3083–9. ©2010 AACR.