Safe lipid nanocapsule-based gel technology to target lymph nodes and combat mediastinal metastases from an orthotopic non-small-cell lung cancer model in SCID-CB17 mice.

The purpose of this study is the assessment of gel technology based on a lauroyl derivative of gemcitabine encapsulated in lipid nanocapsules delivered subcutaneously or intravenously after dilution to (i) target lymph nodes, (ii) induce less systemic toxicity and (iii) combat mediastinal metastases from an orthotopic model of human, squamous, non-small-cell lung cancer Ma44-3 cells implanted in severe combined immunodeficiency mice. The gel technology mainly targeted lymph nodes as revealed by the biodistribution study. Moreover, the gel technology induced no significant myelosuppression (platelet count) in comparison with the control saline group, unlike the conventional intravenous gemcitabine hydrochloride treated group (P<0.05). Besides, the gel technology, delivered subcutaneously twice a week, was able to combat locally mediastinal metastases from the orthotopic lung tumor and to significantly delay death (P<0.05) as was the diluted gel technology delivered intravenously three times a week. FROM THE CLINICAL EDITOR: Lung cancer is one of the leading causes of mortality worldwide. A significant proportion of patients with this disease have lymph node metastasis. In this study, the authors investigated the use of lipid nanocapsules, loaded with the lipophilic pro-drug gemcitabine for targeting tumors in lymph nodes after subcutaneous injection. This delivery method was shown to be effective in controlling tumor progression and may be useful in future clinical use.

[Automated hematology analysers and spurious counts Part 2. Leukocyte count and differential]. / Anomalies et erreurs de détermination de l'hémogramme avec les automates d'hématologie cellulaire Partie 2. Numération et formule leucocytaires.

Using hematology analysers, white blood cell (WBC) counts and differentials (either three or five parameters) may be ascertained after Red Blood Cell (RBC) lysis and analysis using either impedance and/or optical (laser) technology. Cells or particles not destroyed by lytic agents are enumerated as WBC: abnormal particles may be observed on WBC differential scattergrams, if performed, appearing as a variable number of dots, which location may help to ascertain the nature of the abnormality. Spuriously low WBC counts are rare, mainly related to agglutination in the presence of ethylenediamine tetra-acetic acid. Cryoglobulins, lipids, insufficiently lysed RBC, erythroblasts and platelet aggregates are common situations increasing WBC counts. So far, many current high performance analysers clearly identify and enumerate erythroblasts now. In normal patients and in reactive disorders automated differential provides true and accurate results. However, failure to enumerate accurately basophilic granulocytes and monocytes is not uncommon. Using myeloperoxidase cytochemistry to ascertain differential may lead to slide review if the enzyme expression is low or absent. Low number of abnormal cells (blasts, lymphoma cells, dysplastic granulocytes) may be missed, more frequently if leukopenia is present. In many but not all instances flagging and/or an abnormal WBC differential scattergram will alert the operator. Although these flags are sensitive enough to allow the identification of several spurious counts, only the most sophisticated analysers have optimal flagging, whereas more simple ones, especially those without a WBC differential scattergram, do not demonstrate the same sensitivity for the detection of abnormal results.

[Automated hematology analysers and spurious counts Part 3. Haemoglobin, red blood cells, cell count and indices, reticulocytes]. / Anomalies et erreurs de détermination de l'hémogramme avec les automates d'hématologie cellulaire Partie 3. Hémoglobine, hématies, indices érythrocytaires, réticulocytes.

Several situations lead to abnormal haemoglobin measurement or to abnormal red blood cells (RBC) counts, including hyperlipemias, agglutinins and cryoglobulins, haemolysis, or elevated white blood cells (WBC) counts. Mean (red) cell volume may be also subject to spurious determination, because of agglutinins (mainly cold), high blood glucose level, natremia, anticoagulants in excess and at times technological considerations. Abnormality related to one measured parameter eventually leads to abnormal calculated RBC indices: mean cell haemoglobin content is certainly the most important RBC parameter to consider, maybe as important as flags generated by the haematology analysers (HA) themselves. In many circumstances, several of the measured parameters from cell blood counts (CBC) may be altered, and the discovery of a spurious change on one parameter frequently means that the validity of other parameters should be considered. Sensitive flags allow now the identification of several spurious counts, but only the most sophisticated HA have optimal flagging, and simpler ones, especially those without any WBC differential scattergram, do not share the same capacity to detect abnormal results. Reticulocytes are integrated into the CBC in many HA, and several situations may lead to abnormal counts, including abnormal gating, interference with intraerythrocytic particles, erythroblastosis or high WBC counts.

Tax gene expression and cell cycling but not cell death are selected during HTLV-1 infection in vivo.

BACKGROUND: Adult T cell leukemia results from the malignant transformation of a CD4+ lymphoid clone carrying an integrated HTLV-1 provirus that has undergone several oncogenic events over a 30-60 year period of persistent clonal expansion. Both CD4+ and CD8+ lymphocytes are infected in vivo; their expansion relies on CD4+ cell cycling and on the prevention of CD8+ cell death. Cloned infected CD4+ but not CD8+ T cells from patients without malignancy also add up nuclear and mitotic defects typical of genetic instability related to the expression of the virus-encoded oncogene tax. HTLV-1 expression is cancer-prone in vitro, but in vivo numerous selection forces act to maintain T cell homeostasis and are possibly involved in clonal selection. RESULTS: Here we demonstrate that the HTLV-1 associated CD4+ preleukemic phenotype and the specific patterns of CD4+ and CD8+ clonal expansion are in vivo selected processes. By comparing the effects of recent (1 month) experimental infections performed in vitro and those observed in cloned T cells from patients infected for >6-26 years, we found that in chronically HTLV-1 infected individuals, HTLV-1 positive clones are selected for tax expression. In vivo, infected CD4+ cells are positively selected for cell cycling whereas infected CD8+ cells and uninfected CD4+ cells are negatively selected for the same processes. In contrast, the known HTLV-1-dependent prevention of CD8+ T cell death pertains to both in vivo and in vitro infected cells. CONCLUSIONS: Therefore, virus-cell interactions alone are not sufficient to initiate early leukemogenesis in vivo.

[Automated hematology analysers and spurious counts. Part 1. Platelets]. / Anomalies et erreurs de détermination de l'hémogramme avec les automates d'hématologie cellulaire. Partie 1. Les plaquettes sanguines.

Hematology analysers provide now quick, accurate, and reproducible cell blood counts. However, depending on detection methods, spurious counts may occur. If undetected, such spurious counts may lead to inappropriate medical care and to unneeded explorations. Focusing first on platelet counts, situations leading to spurious decrease include several preanalytical considerations, the major one corresponding to EDTA-induced platelet aggregation and to platelet satellitism around polymorphs. In other instances, related to the presence of small particles mimicking platelets, including fragmented red blood cells, lipids, cryoglobulins, fibrin strands, or cytoplasmic fragments of leukocytes, spurious elevation of platelet count may occur. According to the analyser and to the methods used for the determination of the cell blood count, flags or messages related to these spurious changes differ. For each spurious change, the authors describe the mechanism leading to the anomaly, the way the analysers generate flags, and what should be done to provide accurate results.

HTLV-1 propels untransformed CD4 lymphocytes into the cell cycle while protecting CD8 cells from death.

Human T cell leukemia virus type 1 (HTLV-1) infects both CD4+ and CD8+ lymphocytes, yet it induces adult T cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Here we show that in vivo infected CD4+ and CD8+ T cells displayed similar patterns of clonal expansion in carriers without malignancy. Cloned infected cells from individuals without malignancy had a dramatic increase in spontaneous proliferation, which predominated in CD8+ lymphocytes and depended on the amount of tax mRNA. In fact, the clonal expansion of HTLV-1-positive CD8+ and CD4+ lymphocytes relied on 2 distinct mechanisms--infection prevented cell death in the former while recruiting the latter into the cell cycle. Cell cycling, but not apoptosis, depended on the level of viral-encoded tax expression. Infected tax-expressing CD4+ lymphocytes accumulated cellular defects characteristic of genetic instability. Therefore, HTLV-1 infection establishes a preleukemic phenotype that is restricted to CD4+ infected clones.

Hyperdiploidy is a common finding in monoclonal gammopathy of undetermined significance and monosomy 13 is restricted to these hyperdiploid patients.

PURPOSE: Two pathways, hyperdiploid and nonhyperdiploid, are proposed for progression to plasma cell neoplasia. Implication of monosomy 13 (Delta13) is unclear in monoclonal gammopathy of undetermined significance (MGUS), and data on DNA content of plasma cells [DNA index (DI)] are rare. EXPERIMENTAL DESIGN: We ascertained DI in 169 multiple myeloma (MM) and 96 MGUS patients. Interphase fluorescence in situ hybridization (FISH) coupled to cytoplasmic staining of specific Ig (cIg-FISH) was done to look for trisomies and to ascertain Delta13. RESULTS: Hyperdiploidy and hypodiploidy were found in 54% and 11.5% of MGUS patients and in 59.5% and 25% of MM patients, respectively. In MGUS patients tested using probes for odd chromosomes, cIg-FISH showed association between trisomies for chromosomes 3, 7, 9, 11, or 15 and hyperdiploidy. Delta13 was found in 45.3% and 24.6% of MM and MGUS patients, respectively. Most Delta13 cases observed in MGUS were found within hyperdiploid clones, 38% versus 11% in hypodiploid cases, in sharp contrast with the occurrence of Delta13 in MM patients, 31.9% and 76.3%, respectively. That peculiar distribution of Delta13 according to DI persisted with other thresholds used to ascertain hyperdiploidy, such as DI >or= 1.05. A strong relationship between IgA peak and hypodiploidy (P = 0.007) was only observed in MM, whereas lambda light chain was significantly associated with hypodiploidy in MGUS (P = 0.001) and MM (P = 0.05). Hyperdiploidy shows similar pattern in MGUS and MM. CONCLUSION: This fits well a hyperdiploid pathway leading to MM after a preceding MGUS stage. Yet-to-be-determined secondary event(s) needs to occur for the transition to MM, unrelated to changes in chromosome number or to loss of chromosome 13. In contrast, the "nonhyperdiploid" pathway needs to be clarified further because hypodiploidy is less common in MGUS than in MM and Delta13 is rare in hypodiploid MGUS patients compared with hypodiploid MM patients.

[Monoclonal gammopathies of undetermined significance]. / Gammapathies monoclonales de signification indéterminée.

Monoclonal gammopathies of undetermined significance constitute a body of asymptomatic diseases characterized by a selective increase of a single serum immunoglobulin due to an only dysregulated clone of B lymphocytes. They occur in up to 10% of persons over 80 years of age. The diagnosis is suggested in presence of both M component in serum <30 g/L and bone marrow plasma cells <10% by the exclusion of multiple myeloma or lymphoproliferative disorders. In most MGUS, however, populations of normal and myelomatous plasma cells coexist in a stable ratio. The overall relative risk for progression is 7 times the expected rate, depending from the disease and the type of M component.

[Acquired Pelger-Huët anomaly/abnormal chromatin clumping of granulocytes after allogeneic hematopoietic stem cell transplantation for acute myeloid leukaemia: medication or relapse? ]. / Anomalie de Pelger-Huët acquise/condensation anormale de la chromatine des granulocytes après allogreffe médullaire pour leucémie aiguë myéloïde : iatrogène ou signe de rechute ?

An acute myeloid leukemia was diagnosed in a 53-year-old female patient. She received an allogeneic stem cell transplant. After this transplant, some neutrophils with hyposegmented nucleus and abnormal chromatin clumping appeared in the peripheral blood, and their number gradually increased. The hypothesis of early relapse after transplant was ruled out and drug-related anomaly was suspected. The authors discuss about morphological features of constitutional and acquired Pelger-Huët anomaly. In the patient reported here, ciclosporine seemed to be involved in the phenomenon, as the morphological anomaly of the neutrophils gradually decreased after the drug was discontinued.

Irregular nuclear shape of bone marrow plasma cells defines a multiple myeloma subgroup related to hypodiploidy and to short survival.

Morphological changes of plasma cells (PC) are common in multiple myeloma (MM). Loss of round or oval nuclear shape has been related to cell malignancy in human, and we looked for the occurrence of such morphological change on PC from bone marrow (BM) smears in a retrospective series of 169 MM patients at diagnosis. Nuclear shape changes of PC differed according to the patients (notch, dumb-bell, folded or monocytoid appearance), even in the same patient; all subtypes were pooled and defined as PC with irregular nuclear shape (PCIN). A significant number of PCIN (>/=5% of all BMPC) was found at diagnosis in 20.7%. Median survival was of 22 months for patients with >/=5% PCIN, and 41 months for others (p=0.0001). Significant relationship was observed with prognostic parameters related intrinsic malignancy of the tumour process but not with beta-2-microglobulin (b2m). A clear-cut relationship was found also between PCIN and hypodiploidy (p=0.0001), but not with deletion of chromosome 13. This study emphasises the relationship between PCIN, an easy-to-ascertain marker of intrinsic malignancy of the tumour process, and adverse prognosis.

Formalin-fixed and paraffin-embedded nodal non-Hodgkin's lymphomas demonstrate the same chromosome changes as those found in frozen samples: a comparative study using interphase fluorescence in situ hybridization.

Cytogenetic studies in lymphomas classically require fresh or frozen tissue, whereas in many instances only paraffin-embedded biopsies are available. We applied an interphase FISH assay on nuclei extracted from thick paraffin sections to determine accuracy of molecular cytogenetics in such samples. Twenty-three lymphoma samples and 4 reactive lymph nodes were tested with various commercially available DNA probes, and hybridization patterns were compared with those obtained on frozen nuclei counterparts. Successful hybridization with all probes tested was observed for 23/27 (85%) paraffin-embedded tissues and for all (100%) frozen samples, and cut-off levels defining positivity were superimposable for both situations. Chromosome changes were detected in the same way, without any false-positive or false-negative cases. Hybridization signals observed on dewaxed samples were either those classically expected to define the relevant chromosome change or were atypical: all atypical changes could be demonstrated also into nuclei from the frozen counterpart. Moreover, all typical and atypical chromosome changes observed on frozen nuclei were also detected in paraffin-embedded tissues. Our study shows that our interphase FISH assay performed on paraffin-embedded samples is a valuable alternate to conventional methods to ascertain diagnosis of lymphomas as to include patients into therapeutic trials.

[Multiorgan failure after sickle cell vaso occlusive attack: integrated clinical and biological emergency]. / Complications en cascade avec défaillance multiviscérale au cours d'une drépanocytose : gestion coordonnée de l'urgence au laboratoire et au service de réanimation médicale.

We describe the case of a 30-year-old patient, suffering from composite S/ß + sickle cell disease. He was hospitalized following a vaso-occlusive attack with acute bone pains. Despite an analgesic treatment and transfusion of three units of red blood cells, a non-regenerative anemia appeared within 24 hours. One day later an acute chest syndrome with atelectasis of the left lung and desaturation and multi-organ failure occurred and necessitated the patient's intubation and required him to be placed in an artificial coma. A bronchoalveolar lavage was performed, which eliminated pneumonia but proved, after staining with oil red O, many neutral fatty acid microvacuoles in more than 80% of macrophages, suggesting a pulmonary fat embolism. The hypothesis of a bone marrow necrosis causing a pulmonary fat embolism was discussed and confirmed the next day by the characteristic appearance of the bone marrow. A therapeutic protocol associating iteratively bleeding and red blood cells transfusion was administered on the second day with the objective of maintaining haemoglobin S at less than 20% rate. Successive haemoglobin S assay was applied using a high performance liquid chromatography (HPLC) technique with a quick response within one hour after transfusion or bleeding. This protocol resulted in an improvement in the patient's condition, with a gradual normalization of vital signs and extubation twelve days later and discharge without sequelae twenty-five days later. The succession of rare but serious sickle cell complications anaemia which occurred in this patient could be controlled by adapting the laboratory for the clinical emergency.

Routine use of microarray-based gene expression profiling to identify patients with low cytogenetic risk acute myeloid leukemia: accurate results can be obtained even with suboptimal samples.

BACKGROUND: Gene expression profiling has shown its ability to identify with high accuracy low cytogenetic risk acute myeloid leukemia such as acute promyelocytic leukemia and leukemias with t(8;21) or inv(16). The aim of this gene expression profiling study was to evaluate to what extent suboptimal samples with low leukemic blast load (range, 2-59%) and/or poor quality control criteria could also be correctly identified. METHODS: Specific signatures were first defined so that all 71 acute promyelocytic leukemia, leukemia with t(8;21) or inv(16)-AML as well as cytogenetically normal acute myeloid leukemia samples with at least 60% blasts and good quality control criteria were correctly classified (training set). The classifiers were then evaluated for their ability to assign to the expected class 111 samples considered as suboptimal because of a low leukemic blast load (n = 101) and/or poor quality control criteria (n = 10) (test set). RESULTS: With 10-marker classifiers, all training set samples as well as 97 of the 101 test samples with a low blast load, and all 10 samples with poor quality control criteria were correctly classified. Regarding test set samples, the overall error rate of the class prediction was below 4 percent, even though the leukemic blast load was as low as 2%. Sensitivity, specificity, negative and positive predictive values of the class assignments ranged from 91% to 100%. Of note, for acute promyelocytic leukemia and leukemias with t(8;21) or inv(16), the confidence level of the class assignment was influenced by the leukemic blast load. CONCLUSION: Gene expression profiling and a supervised method requiring 10-marker classifiers enable the identification of favorable cytogenetic risk acute myeloid leukemia even when samples contain low leukemic blast loads or display poor quality control criterion.

Plasma cell growth fraction using Ki-67 antigen expression identifies a subgroup of multiple myeloma patients displaying short survival within the ISS stage I.

The current most powerful prognostic model in Multiple Myeloma (MM) combines beta-2 microglobulin (b2m) with albumin, corresponding to the International Staging System (ISS). However, the prognosis of patients within the ISS stage I (high albumin and low b2m) may vary. Ki-67 is a nuclear protein associated with cell proliferation. We retrospectively evaluated the percentage of bone marrow plasma cells expressing Ki-67 antigen (Ki-67 index) in a series of 174 untreated MM patients at diagnosis. Median survival was 51, 41 and 20 months respectively, and median Ki-67 index was 3.0%, 6.1% and 6.5% in ISS stages I, II, and III respectively. Independently of ISS, Ki-67 index > or =4% was highly predictive of adverse prognosis. Ki-67 index correlated with markers of intrinsic malignancy and with markers of tumour burden. Within ISS stage I, median survival was of 31 months (RR of death 2.65) in patients with Ki-67 index > or =4%. Eventually, the combination of Ki-67 with b2m produced an efficient prognostic model, which appeared most effective in our series when compared with b2m and KI-67 with chromosome 13 deletion models. In this series, we demonstrated that a proliferation marker provides clear-cut additional survival prognostic information to b2m into the ISS model.

Genetic abnormalities and survival in multiple myeloma: the experience of the Intergroupe Francophone du Myélome.

Acquired genomic aberrations have been shown to significantly impact survival in several hematologic malignancies. We analyzed the prognostic value of the most frequent chromosomal changes in a large series of patients with newly diagnosed symptomatic myeloma prospectively enrolled in homogeneous therapeutic trials. All the 1064 patients enrolled in the IFM99 trials conducted by the Intergroupe Francophone du Myélome benefited from an interphase fluorescence in situ hybridization analysis performed on purified bone marrow plasma cells. They were systematically screened for the following genomic aberrations: del(13), t(11;14), t(4;14), hyperdiploidy, MYC translocations, and del(17p). Chromosomal changes were observed in 90% of the patients. The del(13), t(11;14), t(4;14), hyperdiploidy, MYC translocations, and del(17p) were present in 48%, 21%, 14%, 39%, 13%, and 11% of the patients, respectively. After a median follow-up of 41 months, univariate statistical analyses revealed that del(13), t(4;14), nonhyperdiploidy, and del(17p) negatively impacted both the event-free survival and the overall survival, whereas t(11;14) and MYC translocations did not influence the prognosis. Multivariate analyses on 513 patients annotated for all the parameters showed that only t(4;14) and del(17p) retained prognostic value for both the event-free and overall survivals. When compared with the currently used International Staging System, this prognostic model compares favorably. In myeloma, the genomic aberrations t(4;14) and del(17p), together with beta2-microglobulin level, are important independent predictors of survival. These findings have implications for the design of risk-adapted treatment strategies.

Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing <2% CD34(+) cells, vs. 1/10.2 for grafts containing > or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.