Gitte Olesen

Chronic neutrophil leukaemia in adolescence and young adulthood

Chronic neutrophil leukaemia (CNL) is a rare myeloproliferative disorder predominantly reported in elderly patients. We present a 15‐year‐old girl and a 25‐year‐old male with CNL. Clonal cytogenetic abnormalities were detected in both patients. One showed trisomy 21 evolving into tetrasomy 21. The second patient showed a unique chromosome aberration during blast crisis: t(2;2)(q32;p24). Both patients were successfully treated with allogeneic bone marrow transplantation (BMT). CNL should also be considered as a differential diagnosis in adolescence and young adulthood. BMT represents a potentially curative treatment option in such patients.

Leukaemia cell drug resistance and prognostic factors in AML

Abstract: In 93 cases of acute myeloid leukaemia (AML) the extent to which prognostic factors mirrored the in vitro cellular chemotherapy resistance (to anthracyclines aclarubicin (Acla) and daunorubicin (Dau) as well as nucleoside analogue cytarabine (Ara‐C)) was investigated using a 4‐d MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide) assay. We found that age at presentation and presence of secondary AML were significantly correlated to leukaemia cell Ara‐C resistance. Thus, analysis of in vitro drug resistance data revealed that age at presentation and presence of secondary leukaemia were both independently correlated to cellular drug resistance, with older age being associated with higher Ara‐C resistance in vitro (p = 0.02 and 0.01 in univariate and multivariate analyses, respectively) and with secondary leukaemia being associated with higher Ara‐C resistance (p = 0.04 and 0.059 in univariate and multivariate analysis, respectively). Median LC‐50 values (Ara‐C) were: 178 ng/ml in paediatric cases, 356 ng/ml in younger adult cases, and 584 ng/ml in elderly (age ≥ 60 yr) cases giving a resistance ratio between these age subgroups of 1:2.0:3.3. Median LC‐50 values (Ara‐C) was 381 ng/ml in de novo cases as opposed to 891 ng/ml (resistance ratio 1:2.3) in secondary cases. By contrast, cytogenetic findings, presenting leucocyte count, FAB‐subtype, and gender were not consistently correlated to in vitro drug resistance to any of the three drugs. We conclude that at least two major adverse prognostic factors in AML (advanced age at presentation and presence of secondary leukaemia) are associated with increased leukaemia cell Ara‐C resistance. High leucocyte count is not associated with increased cellular drug resistance towards Acla, Ara‐C or Dau.

FAB M4 and high CD14 surface expression is associated with high cellular resistance to Ara-C and daunorubicin: implications for clinical outcome in acute myeloid leukaemia.

Abstract: In 145 adult patients diagnosed with non‐M3 acute myeloid leukaemia (AML) the relevance of FAB‐subtype and immunophenotype to in vitro cellular drug resistance towards the anthracyclines aclarubicin (Acla) and daunorubicin (Dau), and the nucleoside analogue cytarabine (Ara‐C), as well as other antileukaemic drugs, was investigated using a 4‐d MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide) assay. We demonstrate that high CD14 expression is highly significantly associated with high cellular Ara‐C and Dau resistance in univariate as well as multivariate analyses. FAB subtypes with highest and lowest cellular Ara‐C resistance were M4 and M5, respectively (P < 0.01, one‐way anova), whereas FAB subtypes with highest and lowest cellular Dau resistance were M4 and M1, respectively (P < 0.01, one‐way anova). By contrast, no significant differences in cellular drug resistance towards Acla could be demonstrated among FAB subtypes. Furthermore, in two cohorts of AML patients treated by two different regimens for remission induction over a period of 15 yr (1985–94, n = 159 and 1995–99, n = 76, respectively) we demonstrate in univariate analyses a significance of CD14 expression with respect to clinical outcome. With the exception of significance to probability of obtaining complete remission in the first cohort (P = 0.03, logistic regression), this significance was, however, lost in multivariate analyses. It was demonstrated that FAB‐M4 patients were older than M5 patients and that high CD14 expression was associated with the presence of secondary AML and older age. We conclude that although cases with high blast cell CD14 expression (and FAB‐M4 cases) were more resistant to Ara‐C as well as Dau in vitro, the clinical and biological significance of this may be debatable because of interactions with major prognostic factors in AML.

Haematopoietic cell transplantation with non-myeloablative conditioning in Denmark: disease-specific outcome, complications and hospitalization requirements of the first 100 transplants

We analysed the outcome and hospitalization requirements of the first 100 patients (Hodgkins disease (HD), N=13; multiple myeloma (MM), N=14; CLL, N=12; non-Hodgkins lymphoma (NHL), N=17; myelodysplastic syndrome (MDS), N=18; AML, N=24 and CML, N=2) treated in Denmark with haematopoietic cell transplantation after non-myeloablative conditioning with TBI 2 Gy±fludarabine. The cumulative incidence of acute GVHD grade II–IV and extensive chronic GVHD was 67 and 49%. After a median follow-up of 534 days, the overall survival, PFS, relapse-related mortality and treatment-related mortality were 59, 50, 25 and 17%, respectively. Patients with CLL, NHL, AML and MDS with <5% blasts at any time had a favourable outcome with a PFS of 61–71%. Patients with MM, HD and MDS and a history of ⩾5% blasts had a less favourable outcome with a PFS of 19–38% (P=0.001). The cumulative incidence of discontinuation of immunosuppression was 37%. During the first and second year post transplant, patients experienced a mean of 41 and 13 outpatient clinic visits, and 53 and 16 days of hospitalization. Sixteen patients were admitted to the intensive care unit, of whom eight are still alive. In conclusion, transplantation outcomes were encouraging, but complications requiring admission and outpatient clinic visits occur frequently post transplant.

Delineation of erythropoiesis in normal and malignant bone marrow using monoclonal antibody AS-E1 directed against transferrin receptors (CD71).

Abstract: We have delineated the erythropoietic compartment in normal and malignant bone marrow (BM) by using the monoclonal antibody (mAb) AS‐E1 directed against the transferrin receptor by flow cytometric (FCM) analysis. In normal BM we found a bimodal expression in antigen density with a minor subset (˜3%) expressing AS‐E1high and a larger subset (˜15%) expressing AS‐E1low. By fluorescence activated cell sorting, morphological examination of smears stained by immunocytochemistry and by BFU–E assays the AS‐E1high fraction was shown to contain cells of erythroid origin (proerythroblasts, basophilic erythroblasts and polychromatic erythroblasts), whereas the AS‐E1low fraction consisted mainly of promyelocytes and myelocytes. In patients with malignant hematological disorders we found a more pronounced heterogeneity in the density and the degree of AS‐E1low expression compared to normal BM, and to further characterize the AS‐E1low cells in patients and to exclude that this broad reactivity interfered with the identification of the AS‐E1high cells, we employed triple‐color FCM assays with mAbs directed against the myeloid surface markers CD13 and CD66 in addition to AS‐E1. In all patients we found that 80–90% of the AS‐E1low cells co‐expressed CD13 and/or CD66 and thus were of myeloid origin. Finally, we evaluated 2 methods for determination of the AS‐E1high subset and found an assay involving forward light scatter and logAS‐E1 density to be sufficient. We conclude that AS‐E1high is a valid FCM marker for the normal erythropoiesis.

Stromal‐mediated down‐regulation of CD13 in bone marrow cells originating from acute myeloid leukemia patients

Abstract: The metallopeptidase CD13 is expressed on normal myeloid cells of monocytic and granulocytic origin and on the surface of leukemic blasts in most acute myeloid leukemias (AML). To study the mechanisms regulating lineage restricted CD13 expression in AML we determined normalised CD13 mRNA levels in bone marrow cells and peripheral blood cells of 27 AML patients. Cells of bone marrow origin had lower levels of normalised CD13 mRNA than cells of peripheral blood origin, even though fluorescence intensity and fraction of cells expressing CD13 on the surface was unchanged. In particular, AML patients with very low levels of normalised CD13 mRNA in bone marrow cells showed an increase in CD13 mRNA expression in peripheral blood. To evaluate the effects of bone marrow microenvironment on CD13 mRNA expression, we cultured leukemic myeloid cells with and without murine stromal cells. Bone marrow cells with high and low CD13 surface expression that entered the stromal layers all down‐regulated CD13 mRNA expression as compared to cells in suspension above. For peripheral blood cells within stromal layers, CD13 mRNA expression was diminished in only 3 out of 6 cases. The ambiguous effect of stromal cells on peripheral blood cells may illustrate a differentiation‐dependent response towards stroma. We determined the polyadenylation status of CD13 mRNA for 9 bone marrow aspirates and 7 peripheral blood samples. Polyadenylation was diminished in bone marrow cells from AML patients with low levels of normalised CD13 mRNA, raising the possibility of involvement of mRNA instability in regulation of CD13 mRNA expression in this subgroup of patients.

Danish patients with untreated multiple myeloma do not harbour human herpesvirus 8

The role of human herpesvirus 8 (HHV‐8) in multiple myeloma (MM) remains controversial. We examined 15 Danish MM patients before cytoreductive therapy. Mononuclear cells isolated from peripheral blood and bone marrow aspirates, as well as long‐term cultured bone marrow stromal cells, were assayed for the presence of HHV‐8 DNA. All material was tested by three simple unnested polymerase chain reaction (PCR) assays (amplifying regions of ORF26, ORFK1 and ORF75) and two nested PCR assays (amplifying regions of ORF26). HHV‐8 was not demonstrated in any of the samples. Our findings do not suggest an association between HHV‐8 and MM in the Danish population.

Long-term culture of hematopoietic stem cells - validating the stromal component of the CAFC assay

BACKGROUND The stroma-based long-term culture is the assay of choice when a functional detection of primitive hematopoietic cells in vitro is sought. However, different stromal cell lines varying in supporting capacity have been raised and applied in different laboratories, resulting in a wide range in published frequencies of LTCIC alternative CAFC. METHODS In order to identify the most suitable stromal source in terms of supportive capacity, reproducibility, and ease of handling, we have compared some of the most commonly employed murine cell lines to human bone marrow stroma in secondary long-term culture set-ups. RESULTS Seeking an approximation to the supportive capacity of human BM stroma we found the FBMD-1 cell line supplemented with G-CSF and IL-3 superior to FBMD-1 cells alone, and to M2-10B4 and Sl/Sl cells. Moreover, in co-cultures of CD34(+) cells and the FBMD-1 line, we found week 5 CAFC content highly reproducible (50.5 +/- 6.66 - 54.6 +/- 7.07/10(4) plated cells, p value > 0.95) and the assay was suitable for inter-individual comparison in a clinical setting. In fact, the week 5 CAFC results were even more reproducible than those of the CFU assays (CV 0.03 for the CAFC assay versus 0.13-0.33 for the CFU assays). On the other hand, when extending the culture period to 8 weeks, the cobblestone area formation was best maintained by human BM stroma and the high reproducibility in CAFC enumeration in cultures supported by the FBMD-1 was lost. DISCUSSION Among the stromal cell sources tested, the FBMD-1 line was found to be superior in terms of ease of handling and week 5 CAFC reproducibility. However, this robustness could not be extended to week 8 CAFC.

Peripheral blood accessory cells modulate committed colony-forming units but not 5-week cobblestone-area-forming cell outgrowth from CD34+ cells.

Abstract: While cellular modulation in vitro of committed hematopoietic stem cell (HSC) growth has been known for some time, less is known about the effect of accessory cells (AC) on the growth of more immature HSC. We have examined the effect of peripheral blood (PB) AC on hematopoiesis by coculturing enriched PB CD34+ cells (> 96% pure) with different quantities of CD34− cells (< 1% contamination) harvested from 10 breast cancer patients. As expected colony growth was predominantly present in the CD34+ fractions, in which colony forming units granulocyte‐macrophage (CFU‐GM) varied between 89–3289/105 (median 1422/105 seeded cells) and week 5 cobblestone area forming cells (CAFC) between 64–1330/105 (median 427/105 seeded cells). Few CFU‐GM (0–27/105 seeded cells) and no week 5 CAFC (0–1/105 seeded cells) were present in the CD34− fractions. The addition of PB CD34− cells to cultures of CD34+ cells resulted in a considerable variation in the cloning efficiency at the CFU‐GM level, and the extent of modulation within the single patient was inconsistent between the different CD34+/CD34− cell mixtures. Overall the stimulatory effect was more pronounced than inhibition and on average the CFU‐GM formation per CD34+ cell seeded increased 3 fold (stimulatory effect ranged between 3–17 fold and decreases between 2–10 fold). In contrast, the cloning efficiency at the week 5 CAFC level of differentiation remained unaffected by the addition of different amounts of CD34− cells (the stimulatory effect was maximally 3‐fold and inhibition 3‐fold). We conclude that while the CFU assay is modulated by the presence of AC, the CAFC assay is more robust and can be employed as a reliable and reproducible tool for HSC measurement.

Evaluation of infliximab as second-line treatment of acute graft versus host disease -validating response on day 7 and 28 as predictors of survival

Several immunosuppressive drugs have been proposed for second-line treatment of steroid-refractory acute graft versus host disease (aGvHD) after allogeneic hematopoietic stem cell transplantation. However, the studies on these drugs are small, retrospective, uncontrolled and use different endpoints. Therefore, it remains unknown which treatment is superior. We retrospectively evaluated 68 consecutive patients treated with infliximab for aGvHD. We adhered to recently proposed guidelines for aGvHD trials and thus evaluated response on day 7 and 28. Furthermore, we assessed the composite endpoint 6 months freedom from treatment failure (6MFTF). The majority of patients had grade III-IV aGvHD. We found that 41 patients (60%) responded on day 7 and 31 patients (46%) on day 28. Twenty-four patients (35%) achieved 6MFTF. The main reasons for failure within 6 months were death (n = 31) or additional immunosuppression (n = 16). By six and 24 months, 44 and 34% of the patients were alive respectively. Patients with response to infliximab on day 7 and 28 had significantly higher overall survival (OS) probability than non-responders. We show that response on day 7 and 28 identifies high and low risk groups. Patients who fail to respond should be identified early and offered alternative therapy.