Comorbidity and performance status in acute myeloid leukemia patients: a nation-wide population-based cohort study.

As the world population ages, the comorbidity burden in acute myeloid leukemia (AML) patients increases. Evidence on how to integrate comorbidity measures into clinical decision-making is sparse. We determined the prognostic impact of comorbidity and World Health Organization Performance Status (PS) on achievement of complete remission and mortality in all Danish AML patients treated between 2000 and 2012 overall and stratified by age. Comorbidity was measured using a modified version of the Charlson Comorbidity Index, with separate adjustment for pre-leukemic conditions. Of 2792 patients, 1467 (52.5%) were allocated to intensive therapy. Of these patients, 76% did not have any comorbidities, 19% had one comorbid disease and 6% had two or more comorbidities. Low complete remission rates were associated with poor PS but not with comorbidity. Surprisingly, among all intensive therapy patients, presence of comorbidity was not associated with an increased short-term mortality (adjusted 90 day mortality rate (MR)=1.06 (95% confidence interval (CI)=0.76-1.48)) and, if any, only a slight increase in long-term mortality (91 day-3 year adjusted MR=1.18 (95%CI=0.97-1.44). Poor PS was strongly associated with an increased short- and long-term mortality (adjusted 90 day MR, PS⩾2=3.43 (95%CI=2.30-5.13); adjusted 91 day-3 year MR=1.35 (95%CI=1.06-1.74)). We propose that more patients with comorbidity may benefit from intensive chemotherapy.

Validation and clinical implication of a quantitative real-time PCR determination of MDR1 gene expression: comparison with semi-quantitative PCR in 101 patients with acute myeloid leukemia.

INTRODUCTION: The multidrug resistance protein 1 (MDR1) has the capacity to extrude chemotherapeutics and has been implicated in treatment failure in acute myeloid leukemia (AML). Previous methods for determination of MDR1 expression have included dye exclusion, demonstration of P-glycoprotein by flow cytometry and/or immunohistochemistry, and molecular polymerase chain reaction (PCR)-based assays for RNA expression. However, these assays have either proven difficult to standardize or tedious to perform. We have therefore designed a real-time quantitative (RQ)-PCR based assay measuring MDR1 gene expression and validated it in AML patients by direct comparison with a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay. PATIENTS AND METHODS: Bone marrow or peripheral blood from 101 AML patients diagnosed (1987-96) at our department were assessed for quantitative expression of MDR1 employing TaqMan RQ-PCR. These data were compared with results obtained by a semi-quantitative competitive PCR assay employing an artificial internal RNA construct. RESULTS: While the RQ-PCR method was able to determine MDR1 gene expression in a continuous fashion over five logs, the semi-quantitative PCR only yielded data in a discontinuous fashion and over four logs at best. Compared with the MDR1 positive and negative cell lines 8226 DOX40 and REH AML cells exhibited variation of 10 PCR cycles, equivalent to a 1000-fold difference. A significant correlation was observed between the two methods, Spearman's correlation coefficient = -0.502, P-value = 10-5. CONCLUSION: We conclude that, RQ-PCR is a novel methodology, which enables sensitive and quantitative measurement of MDR1 gene expression. This assay is moreover suitable because of its high throughput for longitudinal follow-up and large number of patients.

Pretreatment leukaemia cell drug resistance is correlated to clinical outcome in acute myeloid leukaemia.

In 85 adult patients diagnosed with acute myeloid leukaemia (AML) and treated at the same institution during a 5-yr period, the clinical significance of in vitro cellular drug resistance to the anthracyclines aclarubicin (Acla) and daunorubicin (Dau) as well as the nucleoside analogue cytarabine (Ara-C) was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. In 59 patients of whom 40 were treated by the combination of Acla and Ara-C we found that leukaemia cell drug resistance towards Acla was higher (by a factor 2.80) in patients who failed to enter complete remission (CR) after the first cycle of induction chemotherapy as compared to patients who entered complete remission. The relationship was significant in univariate as well as multivariate analysis (p=0.02 and 0.03, respectively). By contrast, no in vitro single drug resistance values were consistently correlated to other parameters of clinical outcome (overall CR rate, overall survival (OS), or continuous complete remission (CCR)), whereas the combined Acla and Ara-C drug resistance profile (Acla/Ara-C DRP) was of prognostic significance to overall survival of all 85 patients (p=0.004) as well as to the CCR of 39 complete responders (p=0.04). These findings remained statistically significant in multivariate analyses correcting for other variables influencing clinical outcome including patient age, leukocyte count, karyotype, FAB-subtype, and presence/absence of secondary AML. We conclude that the in vitro drug resistance of leukaemia cells at time of disease presentation appears to be independent of prognostic significance to short- and long-term clinical outcome in AML.

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.

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.

Many unbalanced translocations show duplication of a translocation participant. Clinical and cytogenetic implications in myeloid hematologic malignancies.

If a translocation is followed by loss of one of the two derivative chromosomes, the result is an unbalanced translocation, showing monosomy for the segments making up the lost derivative. We have found that in most unbalanced translocations, a third event takes place: a morphologically normal copy of one of the two translocation participants is added to the karyotype. This creates a complex abnormal karyotype with monosomy, disomy, and trisomy for different segments of the translocation participants. We have examined 82 unbalanced translocations from 77 patients, 73 of whom had a myeloid hemopoietic malignancy. Acquisition of a normal copy of a translocation participant was found in 49 translocations. Twenty-five of these showed trisomy for 1q. In 16 of the 25 1q-trisomic cases the translocation was t(1;7)(q10;p10) (trisomy for 1q and monosomy for 7q). Patients with trisomy for 1q were younger than the remaining patients. Whereas those with t(1;7))(q10;p10) showed brief survivals, those with trisomy 1q but monosomy for regions other than 7q survived longer than the remaining patients. We conclude that most unbalanced translocations involve a partial trisomy, that 1q is trisomic far more frequently than any other segment, and that partial trisomy is associated with patient age and survival.

Biology of multiple drug resistance in acute leukemia.

Since the early 1970s, multiple drug resistance (MDR) has been known to exist in cancer cells and is thought to be attributable to a membrane-bound, energy-dependent pump protein (P-glycoprotein [P-gp]) capable of extruding various related and unrelated chemotherapeutic drugs. P-gp is coded for by the MDR1 gene, which in the human genome is located on the long arm of chromosome 7 (7q21-31). At the cellular level, the function of P-gp has been extensively investigated in human cancer. Although innumerable reports have been published in which P-gp has been shown to confer MDR to malignant (including leukemia) cells, so far, large-scale studies in the clinical setting have not convincingly proven that MDR1 plays a major role in clinical drug resistance when the influence of other known prognostic factors in human leukemia are taken into account. At present, results from phase 3 clinical trials evaluating the efficiency of inhibiting (or reversing) the function of P-gp in hematologic malignancies are eagerly awaited. Moreover, the horizon of cellular drug resistance in human cancer has during recent years widened dramatically. Thus, an array of different molecules and mechanisms by which resistant cells can escape the cytotoxic effect of anticancer drugs has now been identified. These molecules and mechanisms include apoptosis-related proteins. In this article, we review the different methods for determining MDR and, in particular, methods for determining P-gp/MDR1, with special reference to their potential importance for therapeutic strategies in human acute leukemia.

Leukaemia cell drug resistance and prognostic factors in AML.

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 > or = 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.

Relevance of in vitro leukaemia cell survival to short- and long term clinical outcome in AML.

In ninety-three cases of newly diagnosed acute myeloid leukaemia (AML) we investigated the importance to short- and long term clinical outcome of the in vitro short term leukaemia cell survival as measured by a 4-day MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-assay. In 67 patients treated by intravenous remission induction therapy we found that patients who after the first induction cycle or after induction therapy overall achieved a complete remission (CR) had leukaemia cells with significantly lower in vitro cell survival ability than cells of non-responders (p = 0.02 and 0.06, respectively). These relations remained statistically significant in subsequent multivariate analyses. Likewise, a favourable effect of low in vitro leukaemia cell survival on overall survival of the patients was detected in the (largest) subgroup of adult patients treated uniformly by the same remission induction regimen as well as in all patients. However, in the 44 patients, who achieved CR, the in vitro leukaemia cell survival did not show significance to remission duration or time to first relapse. Furthermore, the leukaemia cell survival (MTT-assay) did not to correlate with the Bcl-2 expression level (quantitative flow cytometry) of the leukaemia cells (r = 0.18, n = 34, p = 0.32). In addition, in a cell line model employing the growth factor dependent MO7 human AML cell line, growth factor withdrawal was associated with rapid onset of cellular apoptosis as evaluated by morphology, occurrence of a subG1 peak in DNA histograms, and loss of cellular activity in the MTT-assay. In contrast, a more moderate decline in Bcl-2 expression and gradual loss of ability to exclude the trypan blue dye was seen in the leukaemia cells in response to growth factor withdrawal. We conclude, that the MTT-assay provides a simple and sensitive method for measuring in vitro cell survival. The differences in leukaemia cell survival seen in AML may well be clinically relevant and may help to provide a better understanding of clinical drug resistance.

Trisomy 10 survival: a literature review and presentation of seven new cases.

Trisomy 10 as the only chromosome aberration is a rare phenomenon in malignant and premalignant hemopoietic disorders. We describe 7 new cases and have found another 12 in the literature. It appears that, whereas adult patients have myeloid disorders (acute myeloid leukemia, myeloproliferative, or myelodysplastic syndromes), in children the diagnosis is lymphocytic leukemia or lymphoma. The median survival was 122 months in the total material. Age above 60 years proved to be a significant adverse factor (median survival only 5 months; p = 0.003). None of the other clinical, cytogenetic, or hematological variables were of demonstrable prognostic importance. In contrast with the larger trisomy 10 clones, those of limited size were associated with nonleukemic diagnoses, normal or slightly elevated leukocyte counts, and few or no circulating blasts. This may suggest that expansion of the trisomy 10 clone is associated with clinical and hematological progression.

MDR1 gene expression and drug resistance of AML cells.

We investigated the cellular drug resistance to aclarubicin (Acla), cytosine arabinoside (Ara-C), daunorubicin (Dau), doxorubicin (Dox), etoposide (Etop) and mitoxantrone (Mitox) using the MTT assay at time of disease presentation in 93 cases of acute myeloid leukaemia (AML). In 31 cases we concomitantly investigated MDR1 (multiple drug resistance 1 gene) expression (semi-quantitative competitive RT-PCR) of the leukaemic cells. Drug resistance towards Dau, Dox and Etop was correlated to the MDR1 expression of the AML cells (P<0.05) with high MDR1 expression being associated with high drug resistance towards these drugs. Although the data did not allow firm conclusions to be drawn on the correlation between MDR1 expression and drug resistance towards Ara-C and Mitox, the drug resistance towards Acla clearly was not correlated to, or dependent on, the MDR1 expression level of the AML blast cells. In addition, when examining the cross-activities among the six drugs distinct patterns emerged. Thus, high to very high degrees of cross-activity were found to exist between Dau, Dox, Etop and Mitox, whereas Ara-C had moderate cross-activity with the other drugs except Acla, which showed absent to moderate cross-activity with the other drugs. We conclude that MDR1 gene expression is of significance for cellular drug resistance towards specific (MDR1-related) drugs in AML, whereas it is not of significance regarding drug resistance towards other drugs, which is the case with the anthracycline Acla. We suggest that in the place of other more or less complicated ways to circumvent MDR1-mediated drug resistance, Acla may be used to replace Dau, Dox and other MDR1-related drugs if proven as potent as the drug it is to substitute.

Relation of blast cell survival and proliferation to chemotherapy resistance in AML.

We have investigated the in vitro blast cell survival (viability) and drug resistance to cytosine arabinoside (Ara-C), daunorubicin (Dau), mitoxantrone (Mitox) and aclarubicin (Acla) of fresh leukaemic blast cells from 80 patients with newly diagnosed acute myeloid leukaemia (AML) employing the semiautomated colourimetric MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-assay. In 15 cases we concurrently investigated the relation between in vitro blast cell survival (MTT assay) and blast cell proliferation (3H-thymidine incorporation) in the presence and absence of myeloid growth factors (GFs) G-CSF, GM-CSF and IL-3 (individually and in combination). A highly significant correlation was found between blast cell survival and blast cell proliferation (r = 0.87, P < 1 x 10(-4). Furthermore, in 40 evaluable adult patients who completed intravenous induction chemotherapy and were evaluable for response to chemotherapy we found a positive correlation between in vitro blast survival (MTT assay) and response to chemotherapy with high blast survival being associated with poor response to chemotherapy (P = 0.05). Moreover, in a multivariate analysis, high blast cell survival was significantly associated with high CD13 expression and monocytic phenotype (P = 0.0003 and P = 0.02, respectively). Furthermore, we found an inverse relationship between the baseline proliferation of the blasts and the magnitude of response to the GFs (P < 0.02), indicating that cells with low baseline proliferation were more readily stimulated by growth factors. Finally, we found a significant correlation between leukaemic cell survival and cellular drug resistance towards Dau (P = 0.001) and Mitox (P = 0.03), but not towards Ara-C (P = 0.68) and Acla (P = 0.13). We conclude that high in vitro leukaemic cell survival is associated with drug resistance in vivo and in vitro, and furthermore is correlated with high blast cell proliferation and some adverse prognostic factors previously identified in AML.

Synergistic and antagonistic effects of myeloid growth factors on in vitro cellular killing by cytotoxic drugs.

The effect of stimulating acute myeloid leukemia blast cells with a combination of growth factors (G-CSF, GM-CSF, and IL-3) on cellular resistance to the antileukemia drugs Ara-C, daunorubicin, aclarubicin, and mitoxantrone was studied. For assessment of in vitro cellular drug resistance the MTT assay was employed. Stimulated cells showed enhanced sensitivity to Ara-C (p < 0.02), whereas a significant increase in cellular drug resistance to daunorubicin (p < 0.02) was observed. Variable and statistically non-significant changes in drug resistance to aclarubicin and mitoxantrone was induced by stimulation of the blast cells. We conclude on the basis of these observations that myeloid growth factors should be used with caution in combination with daunorubicin in AML treatment until further confirmatory evidence has been presented by other investigators.