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.

[Hydroxyurea-induced leg ulcers in patients with chronic myeloproliferative disorders]. / Hydroxyurea-inducerede underekstremitetsulcera hos patienter med kroniske myeloproliferative tilstande.

INTRODUCTION: Hydroxyurea (HU) is usually a well-tolerated antineoplastic agent, which is commonly used in the treatment of myeloproliferative disorders. Dermatological abnormalities are often seen in patients receiving long-term HU therapy. Leg ulcers have been reported occasionally. MATERIAL AND METHODS: We carried out a prospective and descriptive study of patients who developed leg ulcers while receiving HU therapy. RESULTS: Between 1.1.1997 and 1.2.1998, chronic cutaneous leg ulcers were found in five out of a total of 28 patients treated with HU. The average age was 76 years (64-87 years). Two patients had chronic myelogenous leukaemia in a non-accelerated phase, two polycytaemia vera, and one essential thrombocytosis. The average duration of HU therapy was 30 months (10-55 months) before ulcerations appeared. These were typically located on the malleolar and/or perimalleolar area, and were very painful. HU therapy was discontinued and replaced by busulphan or anagrelide. Within 1.5-11 months of discontinuation of the treatment, the wounds had healed or improved. DISCUSSION: We found a surprisingly high number of cutaneous leg ulcers in patients on HU therapy for chronic myeloproliferative disorders. We believe this disabling complication should be given greater attention and recommend that it is included in the description of the side effects of the drug.

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.

[Should fasting prior to ultrasound examination of the gallbladder, the biliary ducts and the pancreas have to include not-drinking?]. / Skal faste før ultralydundersøgelse af galdeblaere, galdeveje og bugspytkirtel omfatte tørst?

From May to September 1991, 49 patients were examined by ultrasound (Picker CS 9000, 3.5 MHz curved array transducer) before and 30 minutes after drinking 150 ml of water. The volume of the gallbladder decreased significantly from a mean of 23.4 ml to 20.1 ml (p = 0.04). No significant change was found either between the visualisation of the gallbladder and bileducts (0.20 > p > 0.10) or the visualisation of the pancreas (0.40 > p > 0.30). However in 10 patients with cholelithiasis, the calculi were only seen in eight patients after drinking water. Thus, it must still be recommended that patients neither eat nor drink for four hours prior to an ultrasound examination of the gallbladder.

[A complication of oleothorax--after 38 years]. / Komplikation til oleothorax--38 år efter.

We present a case-history of a 50 year-old woman who 38 years earlier had been operated for pulmonary tuberculosis by plugging of the pleural cavity with oil (oleothorax). She developed a tumour under the scapula, which it turned out was due to migration of the oil plug to the subscapular area, an unusual complication to oleothorax. Other complications are described.

The etiology and possible treatment of chronic fatigue syndrome/fibromyalgia.

It is suggested that chronic fatigue syndrome/fibromyalgia is caused by virus injury to the calcium channels leading to larger quantities than usual of calcium ions entering the striated muscle cells. Should this be true, then treatment with a calcium antagonist (CA) may possibly be of value.

[Focal fatty liver. A dilemma of differential diagnosis in ultrasonography and CT]. / Fokal fedtlever. Et differentialdiagnostisk dilemma ved ultralydundersøgelse og CT.

Based on four cases and relevant background literature we partly want to draw attention to the occurrence of focal fatty liver (FFL) and its manifold appearances in connection with ultrasonography (US) and computed tomography (CT), and partly to point out possible pitfalls in US-diagnosis of the liver in cases of extensive fatty liver, where the residual foci of normal liver may incorrectly be interpreted as abnormal lesions. US and CT examination of FFL may cause differential diagnostic problems with risk of misinterpretation and erroneous diagnosis, especially if the phenomenon is unknown. In cases of doubt in US examination, where the uncharacteristic findings together with patient information indicate further examinations, additional CT is first of all recommended. If this still brings no clarity to the diagnosis, the clinical condition in every case will prescribe whether to undertake additional CT after a few weeks or immediately carry out US/CT guided liver biopsy. Current as well as formerly presented research shows that FFL is mainly located in the right hepatic lobe, and possible reasons for that are discussed.