Audit of bone marrow aspirates and trephine biopsies in multiple myeloma – a single centre study

We compared 79 simultaneous marrow aspirates and trephine biopsies from multiple myeloma patients for sensitivity, concordance, quality and clinical relevance. A total of 60 examinations had been performed for initial diagnosis, i.e. in cases of suspected myeloma and 19 at follow‐up. Of which, 45 (57%) of trephine biopsies were less than 1.6 cm before processing and 33 (42%) were crushed and/or fragmented. Overall, only 19/79 (24%) of trephine biopsy specimens were of at least 1.6 cm length prior to processing and not disrupted. On the other hand, 75% of aspirates were particulate and satisfactory. Mean time between receipt of a trephine biopsy specimen and issuance of a histopathological report was 9 days. Although 40% of trephine biopsies yielded information that could not be reliably obtained from a bone marrow aspirate such information was in all cases clinically irrelevant or obtainable by non‐invasive means. In all cases where myeloma was detected in a trephine biopsy it was also detected in a simultaneous bone marrow aspirate, if particulate. However, there were four (5%) cases in which myeloma was detected in such aspirates but not in simultaneously taken trephine biopsies. In cases (n = 19) where repeat aspirates/trephine biopsies were taken for surveillance, concordance was found between reported changes in plasma cell ratio. Our data failed to demonstrate any added benefit from routinely performing trephine biopsies after a particulate specimen had been aspirated for the diagnosis or surveillance of myeloma. Furthermore, they suggest that particulate aspirates may be at least as sensitive as trephine biopsies for detecting myeloma.     

Audit of bone marrow aspirates and trephine biopsies in multiple myeloma--a single centre study

We compared 79 simultaneous marrow aspirates and trephine biopsies from multiple myeloma patients for sensitivity, concordance, quality and clinical relevance. A total of 60 examinations had been performed for initial diagnosis, i.e. in cases of suspected myeloma and 19 at follow-up. Of which, 45 (57%) of trephine biopsies were less than 1.6 cm before processing and 33 (42%) were crushed and/or fragmented. Overall, only 19/79 (24%) of trephine biopsy specimens were of at least 1.6 cm length prior to processing and not disrupted. On the other hand, 75% of aspirates were particulate and satisfactory. Mean time between receipt of a trephine biopsy specimen and issuance of a histopathological report was 9 days. Although 40% of trephine biopsies yielded information that could not be reliably obtained from a bone marrow aspirate such information was in all cases clinically irrelevant or obtainable by non-invasive means. In all cases where myeloma was detected in a trephine biopsy it was also detected in a simultaneous bone marrow aspirate, if particulate. However, there were four (5%) cases in which myeloma was detected in such aspirates but not in simultaneously taken trephine biopsies. In cases (n=19) where repeat aspirates/trephine biopsies were taken for surveillance, concordance was found between reported changes in plasma cell ratio. Our data failed to demonstrate any added benefit from routinely performing trephine biopsies after a particulate specimen had been aspirated for the diagnosis or surveillance of myeloma. Furthermore, they suggest that particulate aspirates may be at least as sensitive as trephine biopsies for detecting myeloma.     

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS‐like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3‐ internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3‐ITD AML blasts co‐cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti‐apoptotic effect is mediated through a combination of direct cell‐cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine‐activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal‐mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3‐ITD AML.     

The effect of granulocyte-macrophage colony-stimulating factor on undifferentiated and mature acute myelogenous leukemia blast progenitors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used recently to recruit undifferentiated acute myelogenous leukemia (AML) blasts into the S-phase of the cell cycle and increase the fraction of cells killed by cell cycle-specific drugs. Using three AML blast colony assays combined with a suspension culture (delta assay), we determined the in vitro effect of GM-CSF on mature and undifferentiated AML blast progenitors obtained from bone marrow aspirates of six AML patients. GM-CSF stimulated AML blast colony proliferation at a concentration of 5 ng/ml in the methylcellulose and the agar clonogenic assays in six of six AML marrow samples. However, in the delta assay, which selects for immature AML progenitors, GM-CSF did not affect AML blast colony-forming cells in five of six AML marrow samples at concentrations ranging from 5 to 300 ng/ml. Our data imply that GM-CSF stimulates mature but not undifferentiated AML blast progenitors. It is therefore possible that GM-CSF may not be beneficial as a recruiting agent in most AML patients.     

In situ RHAMM protein expression in acute myeloid leukemia blasts suggests poor overall survival

Treatment options for patients with high-risk acute myeloid leukemia (AML) include high-dose chemotherapy regimens in combination with allogeneic hematopoietic stem cell transplantation, which takes advantage of the donor T-cell-mediated graft-versus-leukemia effect. Together with beneficial responses observed in assays targeted at leukemia-associated antigens (LAA), this encouraged research on cancer vaccines and adoptive cellular therapies in AML. The receptor for hyaluronic acid-mediated motility (RHAMM, CD168) was identified as one of the most promising LAA in AML. Thus far, little is known about in situ expression in leukemic bone marrow blasts or the prognostic role of RHAMM and its interaction partners in AML. We immunohistochemically analyzed the expression and prognostic significance of RHAMM on trephine bone marrow biopsies from 71 AML cases that had been evaluated for cytogenetics and presence of FLT3-internal tandem duplications and NPM1 mutations. Fifty-five patients (77%) were treated with curative intent, while 16 (23%) received the most appropriate supportive care. Twenty of 71 (28%) AML cases were considered RHAMM+. Receiver operating characteristic curves showed significant discriminatory power considering overall survival (OS) in AML patients treated curatively for RHAMM (p = 0.015). Multivariable analysis revealed that expression of RHAMM in >5% of leukemic blasts identifies a subgroup of curatively treated cases with adverse OS independent of failures to achieve complete remission. RHAMM not only represents a promising LAA with specific T-cell responses in AML but, if assessed in situ on blasts, also a probable prognostic factor.     

Fragment filtration: a method for the accurate determination of flow cytometric kinetic data from bone marrow aspirates

The extent to which bone marrow obtained by conventional aspiration is contaminated by peripheral blood has been confirmed and quantitated. In marrow aspirates from normal subjects the median percentage of nucleated cells that had originated from the peripheral blood was 32% (range 2.5%-64%), in patients with acute leukemia 23% (range 0.5%-96.5%), in patients with chronic leukemia 59% (range 17%-76%), and in patients with lymphoma 31% (range 0.5%-74%). Flow cytometric (FCM) DNA analysis of conventional marrow aspirates from a range of subjects significantly underestimated the proportions of S-phase cells present, when compared with results from trephines obtained at the same time. Having shown, using 51Cr-labeled red cells in mice, that circulating red cells do not reenter the marrow parenchyma, a mathematical correction for contaminating blood similar to that described by Holdrinet et al. was devised. This correction improved the S-phase cell estimate from aspirated marrows, and the corrected values were not significantly different from values from paired trephine samples. A previously described technique for collecting fragments by filtration of aspirated marrow has been adapted for FCM analysis as a more direct way of overcoming problems due to blood contamination. This method was shown to yield estimates of S-phase cells not significantly different from those in paired marrow trephines and offers an alternative to routine trephine biopsies for FCM analysis of marrow cell kinetics.     

Early allogeneic blood stem cell transplantation after modified conditioning therapy during marrow aplasia: stable remission in high-risk acute myeloid leukemia

Two patients with high-risk acute myeloid leukemia (AML) whose bone marrow aspirates showed more than 25% blasts between 2 and 4 weeks after the first induction chemotherapy immediately received modified conditioning therapy with intravenous busulfan at 50% of the usual dose and fludarabine, before hematologic recovery occurred. Unmanipulated G-CSF mobilized peripheral blood stem cells from an HLA-identical sibling donor were transfused and haematopoietic recovery was achieved in both recipients. Both of them are in continuing hematological remission with full donor chimerism 12 and 22 months after transplantation. Early treatment intensification with allogeneic cell therapy during marrow aplasia might cure high-risk AML patients who are unlikely to achieve remission with conventional chemotherapy protocols.     

The challenging task of enumerating blasts in the bone marrow

Enumeration of blasts in the bone marrow is critical for diagnostic, prognostic, and therapeutic response evaluation in myelodysplastic syndromes, myeloproliferative neoplasms and acute leukemias. However, few studies have examined the accuracy and precision of marrow blast counting using standard microscopic procedures. In our study, 4 experienced hematopathologists evaluated blast percentages in marrow using either differential counts on aspirate smears or visual estimates on CD34-stained trephine biopsies. Results of an independent observer's manual counts of individual labeled and unlabeled cells performed on high resolution digital images of CD34-stained trephine biopsies were designated as the “Digital Reference.” Hematopathologists’ blast counts showed excellent interobserver reproducibility, but the counts in smears and trephine biopsies correlated poorly with each other. Compared to the Digital Reference, both smear and trephine evaluations showed positive bias and high variability. The biopsy showed less variability but higher positive bias relative to the smears, indicating that counts were overestimated more in the hematopathologists’ biopsy evaluation. Flow cytometric counts correlated well with the Digital Reference, and cases with high blast count generally showed worse cytogenetic findings. Our results demonstrate the need for better counting methods if significant decisions are made based on microscopic enumeration of blasts. Further efforts should be made to develop markers to better define blast cells and perhaps incorporate automated digital imaging technologies to enumerate them. Also, consideration should be given to quantifying blasts per marrow area in biopsies instead of per nucleated cells.     

Efficient lentiviral transduction of primary human acute myelogenous and lymphoblastic leukemia cells

BACKGROUND AND OBJECTIVES: Gene manipulation and cell vaccines represent innovative strategies to enhance the immunogenicity of cancer cells. We adopted a defective lentivirus derived from the human immunodeficiency virus (HIV)-1 backbone and carrying the enhanced green fluorescent protein (EGFP) gene to transduce primary human acute myelogenous leukemia (AML) and B-precursor acute lymphoblastic leukemia (ALL) cells. DESIGN AND METHODS: AML blasts were maintained with or without cytokines (stem cell factor, FLT3 ligand and interleukin 3) for 48 hours, and successively infected with two spin infection cycles. ALL blasts were cultured on a murine S17 stromal cell line. RESULTS: As regards AML cells, the efficiency of infection at 7 days varied from 8.4 to 37%. As confirmed by cell cycle analysis, cells were, in most of the cases, blocked in different phases of the cycle and did not proliferate during culture: the infection was therefore obtained in the absence of cell proliferation. In contrast, the maintenance of optimal cell viability was of fundamental importance for obtaining good infection levels. As regards ALL blasts, the percentages of infection after 3 days varied from 4.4 to 21%. INTERPRETATION AND CONCLUSIONS: These preliminary data suggest that gene delivery into primary human AML and B-precursor ALL cells by an HIV-1 derived lentiviral vector could represent a strategy for engineering leukemic cells for use as cancer vaccines.     

Proliferation patterns in acute myeloid leukemia: leukemic clonogenic growth and in vivo cell cycle kinetics

Summary In a prospective study of 33 newly diagnosed patients with acute myeloid leukemia (AML), we analyzed the relationship of proliferation parameters with clinical parameters, response to induction therapy, and survival. The median follow-up was 26 months. The proliferative capacity of the leukemic progenitor cells was studied using colony-forming assays (number of colonyforming units, growth pattern, and spontaneous clonogenic growth capacity). The cell kinetic parameters of the bone marrow blasts were determined by in vivo labeling with iododeoxyuridine and subsequent flow cytometry: labeling index (LI), DNA synthesis time (T_s), potential doubling time. No or only weak relationships were observed between the experimental and clinical parameters such as age, sex, % blasts, white blood cell count, FAB subtype, cytogenetics, and % CD 34⁺ cells. This suggests that clonogenic growth and cell cycle kinetics of bone marrow blasts are independent cell biologic properties of AML. No association between the proliferation parameters and induction response rate was noticed. Analysis of the overall survival and event-free survival revealed trends to longer survival rates in patients with a belowmedian LI (7.6%) and below-median T_s value (14.3 h). These trends were more pronounced in the group of de novo AML (n=23), where the prolonged event-free survival in patients with below-median T_s reached statistical significance (p=0.02). None of the other parameters appeared significantly correlated with survival, although there was a trend to longer survival rates in patients who had no spontaneous clonogenic growth capacity (p=0.13). In conclusion, proliferation parameters in leukemic cells provide additional information on the cell biologic characteristics of AML, and these parameters may have prognostic value for response and duration of survival in AML.     

Recovery of drug sensitivity by MS-209, a new multidrug resistance-reversing agent,on acute myelogenous leukaemic blasts and K562 cells resistant to adriamycin cell line

Abstract: The efficacy of MS-209, a quinoline derivative synthesized as a new multidrug resistance (MDR)-reversing agent, was studied on blast cells from 33 acute myelogenous leukaemia (AML) patients and on the human myelogenous leukaemia K562 cell line resistant to adriamycin (K562/ADM). By the addition of MS-209, the intracellular daunorubicin (DNR) contents which had been found to be low in P-gp-positive AML blasts and in K562/ADM were significantly enhanced to the level of P-gp-negative blasts and that of sensitive K562. The intracellular rhodamine (Rh123) contents also increased in P-gp-positive blasts and K562/ADM cells with MS-209. A leukaemic blast colony assay also demonstrated the effect of MS-209, i.e. a high D₁₀ value for DNR of P-gp-positive blasts was reduced to the D₁₀ level similar to that observed in P-gp-negative blasts by the addition of MS-209. The greater DNR sensitivity reversing effect of MS-209 was observed in blasts with higher P-gp positivity. These findings suggest the potential usefulness of MS-209 in overcoming MDR in AML patients, especially those with high P-gp expression. This study clarified the relationship between the clinical outcome of the patients and the P-gp positivity, intracellular DNR content and DNR drug sensitivity of leukaemic progenitors.     

Receptor for platelet-activating factor (PAF) is not detectable by flow cytometry on the surface of myeloid leukemic cells

Flow cytometry was applied to test for platelet-activating-factor receptor (PAF-R) presence on the membranes of acute myeloid leukemia (AML) cells. We have used six human AML cell lines and freshly taken density gradient separated blasts from the bone marrow of ten AML patients covering the majority of French–American–British (FAB) subtypes. Additionally, we have used one histiocytic lymphoma cell line and mature human granulocytes/monocytes as controls. Our results indicate lack of membrane PAF-R on AML of all FAB subtypes tested. This was particularly true for the more mature and differentiated subtypes M₄ and M₅, including monocytic cell elements, and the promyelocytic M₃ AML. In contrast, membrane PAF-R could be easily detected in a histiocytic lymphoma cell line and mature granulocytes/monocytes from peripheral blood used as positive controls. In conclusion, this observation precludes the use of membrane PAF-R as an immunophenotypic marker for AML classification or detection of minimal residual disease.     

Contribution of flow cytometric immunophenotyping and bone marrow trephine biopsy in the detection of lymphoid bone marrow infiltration in non-Hodgkin's lymphomas

The bone marrow (BM) is a frequent site of involvement in non-Hodgkińs lymphomas (NHL) and evidence of an infiltrated BM may implicate different therapeutical regimens. Flow cytometric immunophenotyping of bone marrow aspirates now is included in the assessment of patients with NHL and used as an adjunct to morphologic evaluation in the staging of lymphoma. The aim of the study was to compare flow cytometric immunophenotyping of BM and paraffin section staining of BM biopsies in the marrow involvement of NHL. Cytometric immunophenotyping of bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in 53 B- and T-cell lymphoma patients were performed. We used the following fluorochrom conjugated monoclonal antibodies specific for: CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD23, CD79B, FMC7 and Ig kappagamma light chain. Unilateral BM trephine biopsies were obtained in all cases, fixed, decalcified and paraffin-embedded. Morphologic marrow involvement by lymphoma was found in 24 cases; flow immunophenotyping identified 26 cases with NHL: morphology-positive/flow-positive (n=21), morphology positive/flow-negative (n=3), morphology-negative/flow-positive (n=4), and morphology-negative/flow-negative (n=23). The concurrence rate of BM trephine biopsy and flow cytometric immunophenotyping in evaluation of NHL bone marrow infiltration was 88.7%. Immunophenotyping of the bone marrow of NHL patients by flow cytometry is helpful for assessment of bone marrow infiltration, especially in B-cell disorders. Both trephine biopsies and flow cytometry are better than single investigation for detection of infiltration in NHL.     

Bone marrow cell count per cubic millimeter bone marrow: a new parameter for quantitating therapy-induced cytoreduction in acute leukemia

A new technique is introduced for determining the number of bone marrow cells per cubic millimeter marrow, providing an accurate and objective means for quantitating therapy-induced cytoreduction. The method requires a correction for admixed peripheral blood in bone marrow aspirates to measure the fraction of remaining pure marrow. While cell kinetic differences between blood, aspirates, and biopsies identify the proportion of contaminating blood cells, the ratio of red cell hematocrits in blood and aspirate gives the volume of trapped blood. By combining both procedures, bone marrow cell counts per unit volume pure marrow result (BMC/cu mm BM), which were found highly reproducible. Blast cell counts (BMBC/cu mm BM) were obtained by additional morphological differentiation. BMC and BMBC/cu mm BM were monitored in 16 patients with acute nonlymphoblastic leukemia treated with daunorubicin, cytosine arabinoside, and 6-thioguanine in combination and in 4 patients with end-stage acute leukemias and non-Hodgkin's lymphomas during high-dose thymidine therapy. Total and daily therapy- induced cytoreduction rates were significantly greater (P less than 0.01) in responders than nonresponders to either regimen. Changes in BMC/cu mm BM were also found representative for changes in BMBC/cu mm BM, since the majority of bone marrow cells were blasts. In acute leukemia. BMC/cu mm BM thus provides accurate and objective measurements of treatment efficacy in vivo and after short periods of drug exposure. Differences in cytoreduction rates within the group of responders also suggest possible prognostic implications.     

Granulocyte colony-stimulating factor (G-CSF) receptors on acute myeloblasts leukaemia cells and their relationship with the proliferative response to G-CSF in clonogenic assay

The number and the affinity of granulocyte colony-stimulating factor (G-CSF) receptors expressed by blast cells in acute myeloblastic leukaemia (AML) were determined using radiolabelled recombinant human G-CSF (rhG-CSF). Eighteen of 20 patients demonstrated specific binding, and Scatchard analysis revealed a single class of high affinity (Kd 15-130 pM) G-CSF receptors on the AML blasts. The number of G-CSF receptors varied from 55 to 1,200 per cell (mean 278). In the remaining two patients, specific binding was not observed. The number of G-CSF receptors did not differ significantly between various AML subtypes, but the mean receptor number was the highest on type M2 blasts. A chemical cross-linking study revealed that the G-CSF receptor has an approximate molecular weight of 140,000. Autoradiography showed heterogeneity of the distribution of G-CSF receptors on the AML blasts obtained from a single patient. The number of colonies stimulated by the addition of rhG-CSF varied from 0 to 566 per dish, and blast colony formation was observed in eight of 20 patients. The population mean of G-CSF receptor number expressed by blasts that formed colonies on stimulation with rhG-CSF was significantly higher than that on blasts which did not form colonies. These results suggest that a proliferative response of AML blasts to G-CSF may be predicted when the blasts express a large number of G-CSF receptors. Accordingly, it may be safer to restrict the clinical use of G-CSF to AML patients who have blasts with a low G-CSF receptor expression and no response to G-CSF in blast colony assay.     

Interleukin-1 stimulates proliferation of acute myeloblastic leukemia cells by induction of granulocyte-macrophage colony-stimulating factor release

In this study, we further established the role of interleukin-1 (IL-1) alpha and IL-1 beta as regulators of proliferation of acute myeloid leukemia (AML) cells. IL-1 stimulated tritiated thymidine (3H-TdR) uptake of AML cells in 13 of 28 cases. Cytogenetic analysis confirmed the leukemic clonality of the IL-1-stimulated cells. Most likely, IL-1 exerted these stimulative effects directly on AML blast cells because IL-1 effectively induced 3H-TdR uptake of CD34-positive AML blasts (separated following cell sorting). Furthermore, adherent cell-depleted AML samples of three patients were more effectively stimulated than nondepleted AML fractions. Cluster and colony formation from adherent cell depleted AML samples could also be stimulated with IL-1, ie, in seven of ten cases analyzed. Subsequent experiments indicated that IL-1 stimulation depended on the release of GM-CSF because (1) induction of DNA synthesis of AML cells by IL-1 could be abrogated with antigranulocyte-macrophage colony-stimulating factor (GM-CSF) antibody, (2) conditioned media (CM) prepared from IL-1 stimulated AML blasts (adherent cell depleted) could stimulate the proliferation of purified normal bone marrow progenitors whereas supernatants from nonstimulated AML blasts did not, and (3) GM-CSF was demonstrated in IL-1/AML-CM with a specific radioimmunoassay, while GM-CSF was not detectable in nonstimulated supernatants. In one case of AML showing significant 3H- TdR uptake in the absence of CSFs, this spontaneous DNA synthesis was found to depend on autocrine IL-1 beta release as it could be suppressed with anti-IL-1 beta antibody or anti-GM-CSF. The blockade by anti-IL-1 beta could be overcome by the addition of high concentrations of IL-1 beta as well as GM-CSF. Thus, in this particular case, endogenously produced IL-1 beta had stimulated the release of GM-CSF which resulted in GM-CSF-dependent proliferation. The results indicate that GM-CSF production by AML blasts is often regulated by IL-1 rather than being constitutive.     

Role of the microenvironment in promoting angiogenesis in acute myeloid leukemia

Angiogenesis is a crucial event in the survival and progression of solid tumors. To determine whether angiogenesis in acute myeloid leukemia (AML) is an intrinsic property of leukemic cells, the vascularity of bone marrow biopsies was determined. Bone marrow vascularity in newly diagnosed or post-chemotherapy AML patients was increased 4-fold (P < 0.01) and 8.7-fold (P < 0.01), respectively, relative to controls. Vascular endothelial growth factor (VEGF) expression by AML blast cells was assessed by immunohistochemistry, and bone marrow cell supernatants were assayed for secretion of VEGF, fibroblast growth factor-2 (FGF-2), and endostatin by enzyme-linked immunosorbent assay. Diffuse cytoplasmic and strong extracellular VEGF immunoreactivity was seen in bone marrow aspirates from AML patients, but not controls. In contrast, there was no difference in the levels of VEGF, FGF-2, and endostatin secreted by mononuclear cells cultured from bone marrows of AML patients compared to normal controls following two days of culture in vitro. Total angiogenic potential of bone marrow cell supernatants was assessed by endothelial sprouting in vitro and by a chick chorioallantoic membrane assay. No differences were found between 2-day conditioned medium from normal and AML bone marrow mononuclear cells in either assay. Our data show a discrepancy between bone marrow vascularity and VEGF expression in vivo and VEGF expression and angiogenesis from 2-day conditioned medium ex vivo. This suggests that angiogenesis in AML likely represents a response to microenvironmental factors in vivo, rather than being an intrinsic property of leukemic cells.