Perls' Stain Guidelines from the French-Speaking Cellular Hematology Group (GFHC).

In order to standardize cellular hematology practices, the French-speaking Cellular Hematology Group (Groupe Francophone d'Hématologie Cellulaire, GFHC) focused on Perls' stain. A national survey was carried out, leading to the proposal of recommendations on insoluble iron detection and quantification in bone marrow. The criteria presented here met with a "strong professional agreement" and follow the suggestions of the World Health Organization's classification of hematological malignancies.

Flow cytometry detection of CD138 expression continuum between monotypic B and plasma cells is associated with both high IgM peak levels and MYD88 mutation and contributes to diagnosis of Waldenström macroglobulinemia.

BACKGROUND: Differential diagnosis of Waldenström macroglobulinemia (WM) with other indolent B-cell malignancies is still a challenge. Here, we propose an original and simple analysis of routine flow cytometry (FCM) unraveling the characteristic ongoing plasma cell (PC) differentiation of WM tumor B-cells. METHODS: FCM analysis of both B-cells and PC was performed on a series of 77 patients with IgM peak. MYD88 and CXCR4 mutations were studied using an allele-specific PCR and by high throughput sequencing. RESULTS: Twenty seven (35%), 46 (58%) and 4 (5%) patients were classified as WM, IgM monoclonal gammopathy of undetermined significance (MGUS) or other B-NHL respectively. MYD88 mutation was found in 25/27 WM (93%) and in 29/46 MGUS (63%). Using FCM, monotypic B-cells were found in 27/27 WM (100%) and 34/46 MGUS (74%). Monotypic CD138pos/CD38pos PCs were detected in 23/27 WM (85%) and 25/46 MGUS (54%). Highlighting the ongoing PC differentiation of WM tumor B-cells by FCM, we evidenced a CD138 expression continuum between monotypic B-cells and PCs. This pattern remained absent in control samples and was significantly associated with higher IgM peaks (p = 6.10-5 ) and MYD88 mutations (p = 10-3 ) in both WM and MGUS cases. CONCLUSIONS: FCM exploration of both B-cells and PC led to identify a CD138 expression continuum as an objective marker of ongoing PC differentiation of WM tumor cells and was strongly associated with increased IgM peak levels and MYD88 mutations. This approach could contribute to place FCM at the forefront of WM diagnosis.

Harmonisation of full blood count reports, recommendations of the French-speaking cellular haematology group (GFHC).

AIMS: To propose recommendations related to the presentation, content and formulation of full blood count analysis reports. METHODS: Strong professional agreement among a group of experts from the French-Speaking Cellular Haematology Group (GFHC) was obtained. RESULTS: The following two proposals emerged from the consensus: (1) stratification of comments into three parts upon the discovery of an anomaly in blood cell analysis and (2) selection and/or redefinition of the terms recommended for designating the cell types found in normal and pathological peripheral blood. CONCLUSIONS: The recommendations can help biologists who are currently undergoing the process of accreditation.

Prognostic impact of day 15 blast clearance in risk-adapted remission induction chemotherapy for younger patients with acute myeloid leukemia: long-term results of the multicenter prospective LAM-2001 trial by the GOELAMS study group.

Early response to chemotherapy has a major prognostic impact in acute myeloid leukemia patients treated with a double induction strategy. Less is known about patients treated with standard-dose cytarabine and anthracycline. We designed a risk-adapted remission induction regimen in which a second course of intermediate-dose cytarabine was delivered after standard "7+3" only if patients had 5% or more bone marrow blasts 15 days after chemotherapy initiation (d15-blasts). Of 823 included patients, 795 (96.6%) were evaluable. Five hundred and forty-five patients (68.6%) had less than 5% d15-blasts. Predictive factors for high d15-blasts were white blood cell count (P<0.0001) and cytogenetic risk (P<0.0001). Patients with fewer than 5% d15-blasts had a higher complete response rate (91.7% vs. 69.2%; P<0.0001) and a lower induction death rate (1.8% vs. 6.8%; P=0.001). Five-year event-free (48.4% vs. 25%; P<0.0001), relapse-free (52.7% vs. 36.9%; P=0.0016) and overall survival (55.3% vs. 36.5%; P<0.0001) were significantly higher in patients with d15-blasts lower than 5%. Multivariate analyses identified d15-blasts and cytogenetic risk as independent prognostic factors for the three end points. Failure to achieve early blast clearance remains a poor prognostic factor even after early salvage. By contrast, early responding patients have a favorable outcome without any additional induction course. (ClinicalTrials.gov identifier NCT01015196).

Polycythemia vera transforming to acute myeloid leukemia and complex abnormalities including 9p homogeneously staining region with amplification of MLLT3, JMJD2C, JAK2, and SMARCA2.

Polycythemia vera (PV) is a clonal stem cell disorder characterized by an excessive erythrocyte production. At diagnosis, a normal karyotype is found in < or =80% of cases, but an abnormal karyotype frequently develops with evolution. Trisomy 9 and gains on 9p are some of the most frequent cytogenetic abnormalities, together with trisomy 8 and del(20q) in both PV and idiopathic myelofibrosis. We report the case of a 54-year-old man whose disease was classified as an acute myeloid transformation of PV. Cytogenetic and multicolor fluorescence in situ hybridization (FISH) analysis detected several chromosomal abnormalities that included an amplification of 9p. Complementary FISH analysis established amplification of the 9p22 approximately p24.3 region including several known genes: MLLT3 (alias AF9), JMJD2C (alias GASC1), JAK2, and SMARCA2 (alias BRM). JAK2(V617F) mutation status was quantitatively assessed by allele-specific quantitative polymerase chain reaction. Although crossing points analysis showed JAK2(V617F) mutated alleles at 52%, it is still impossible to describe conclusively the mutational status of the amplified JAK2 gene within the sole homogeneously staining region, because total genomic DNA was extracted for the analysis and not only DNA from cells with the homogeneously staining region. Gains on 9p being among the most common anomalies in PV, amplification of a gene or genes on this region may play a crucial role in the pathogenesis or evolution of PV.

Confirmation of a novel recurrent association: BCR-ABL t(9;22) and t(19;21).

Association of a t(9;22)(q34;q11), BCR/ABL-positive, with a dic(19;21)(p13;p13) has been described in acute lymphoblastic leukemia in relapse, raising the question of whether this association is recurrent. Described here are two cases, one of myeloproliferative disease and one of acute lymphoblastic leukemia, both presenting a masked t(9;22) and t(19;21). Chromosomal rearrangements were ascertained by fluorescence in situ hybridization (FISH) using locus-specific probes, multicolor FISH, and bacterial artificial chromosome array. These additional observations suggest a nonrandom association.

In vivo expression pattern of MICA and MICB and its relevance to auto-immunity and cancer.

Non-conventional MHC class I MIC molecules interact not with the TCR, but with NKG2D, a C-type lectin activatory receptor present on most NK, gammadelta and CD8(+) alphabeta T cells. While this interaction is critical in triggering/calibrating the cytotoxic activity of these cells, the actual extent of its in vivo involvement, in man, in infection, cancer or autoimmunity, needs further assessment. The latter has gained momentum along with the reported expansion of peripheral CD4(+)CD28(-)NKG2D(+) T cells in rheumatoid arthritis (RA). We first initiated to extend this report to a larger cohort of not only RA patients, but also those affected by systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). In RA and SS, this initial observation was further tested in target tissues: the joint and the salivary glands, respectively. In conclusion and despite occasional and indiscriminate expansion of the previously incriminated T cell subpopulation, no correlation could be observed between the CD4(+)CD28(-)NKG2D(+) and auto-immunity. Moreover, in situ, the presence of NKG2D matched that of CD8(+), but not that of CD4(+) T cells. In parallel, a total body tissue scan of both MICA and MICB transcription clearly shows that despite original presumptions, and with the exception of the central nervous system, both genes are widely transcribed and therefore possibly translated and membrane-bound. Extending this analysis to a number of human tumors did not reveal a coherent pattern of expression vs. normal tissues. Collectively these data question previous assumptions, correlating a tissue-specific expression/induction of MIC in relevance to auto-immune or tumor processes.

Telehematology: a pilot experience of cytological diagnosis of acute myeloid leukemia via the Internet. A GOELAMS study.

Although modern communication technology is well developed, telehematology does not readily lend itself to practical laboratory use. Multicenter therapeutic protocols may offer preferential opportunities. The cytologists of the AML-2001 protocol established an innovative organization to demonstrate the reliability of the diagnostic assessment of acute myeloid leukemia through a rapid and decentralized exchange of information via the internet and to define the conditions optimizing expert diagnosis. Telediagnosis appears to be a powerful tool for cytological review and other issues.

Cytogenetic studies of a series of 43 consecutive secondary myelodysplastic syndromes/acute myeloid leukemias: conventional cytogenetics, FISH, and multiplex FISH.

We report a series of 43 consecutive therapy-related myelodysplastic syndromes (t-MDS) or acute myeloid leukemias (t-AML) observed for 6 years. This series consisted of 26 women and 17 men, ages ranging from 9 to 85 years. These cases were classified into three groups according to the primary diagnosis. Conventional cytogenetic and fluorescent in situ hybridization (FISH)/ multiplex FISH (M-FISH) methods were used to analyze cytogenetic characteristics of secondary MDS/AML. The features of chromosomal abnormalities were linked to the nature of the therapy and protocols used. A considerable proportion of recurrent balanced translocations characterized t-AML secondary to therapy. FISH techniques showed that conventional cytogenetics often underestimated associated translocations; some deletions were in fact derivative chromosomes associated with deletions. After treatment for lymphomas and chronic myeloproliferative diseases, there were more complex unbalanced abnormalities than the control group. Compared to other series, recurrent translocations appeared to be more numerous (25%), probably reflecting an evolution of therapeutic modalities.