Inflammatory myoglandular colorectal polyps: a series of seven cases and review of literature.

INTRODUCTION: Inflammatory myoglandular polyp is an unusual but distinct, non-neoplastic type of colorectal polyp, commonly with a distal localization at the recto-sigmoidian level. It was first described in 1992 by Nakamura and his colleagues and it is considered to have few particular histological features. MATERIAL AND METHODS: We report a series of seven cases (two male and five female patients) of myoglandular polyps with different localization from 15 to 40 cm from anus. Only four out of seven cases presented with rectal bleeding, the others polyps we incidentally discovered. RESULTS: The polyps varied between 4 and 30 mm in the maximum diameter. Grossly, they had firm consistency and smooth reddish surface. Histological examination of the specimens revealed hyperplastic glands with occasional cystic dilatation, proliferation of smooth muscle with no regular distribution, a variable amount of granulation tissue (usually minimal) and no evidence of epithelial dysplasia. All the lesions were removed endoscopically without any complications. CONCLUSIONS: Inflammatory myoglandular polyps are distinct histopathological entities, with insufficiently investigated pathogenesis that can include local trauma, mucosal prolapse or ischemia. Being benign they can be removed endoscopically, surgical treatment being reserved in selected cases.

Medullary thyroid carcinoma identified within the first year of life in children with hereditary multiple endocrine neoplasia type 2A (codon 634) and 2B.

CONTEXT: Early prophylactic thyroidectomy in patients with multiple endocrine neoplasia (MEN) type 2 offers the best chance for a normal life expectancy. OBJECTIVE: To analyze the results of thyroidectomy performed during the first year of life in six patients with MEN 2A (codon 634) or MEN 2B (codon 918) syndrome. DESIGN AND SETTING: A university hospital-based prospective study from 2001 to 2008. SUBJECTS AND METHODS: Six family members affected either by MEN 2A (n=3) or MEN 2B (n=3) syndrome were identified through neonatal genetic screening. RESULTS: Total thyroidectomy was performed at a median age of 0.8 year in the six patients, with central lymph node dissection in five. Bilateral millimetric medullary thyroid carcinoma (MTC) was found in all patients, with a unilateral lymph node micrometastasis in two of the three MEN 2B patients. Before thyroidectomy, MEN 2B patients had much higher basal serum calcitonin levels than those with MEN 2A and controls. After thyroidectomy, with a median follow-up of 3.3 years, the six patients had no evidence of persistent MTC. CONCLUSION: Bilateral millimetric MTC may be present during the first year of life in these patients, with lymph node metastases also occurring in MEN 2B patients. These results support a total thyroidectomy at the age of about one year in MEN 2A (codon 634) children with an abnormal serum calcitonin level, and a total thyroidectomy with central neck dissection within the first weeks of life in MEN 2B patients.

Does the RET variant G691S influence the features of sporadic medullary thyroid carcinoma?

OBJECTIVE: The RET (rearranged during transfection) proto-oncogene G691S variant is over-represented in the germline of patients with sporadic medullary thyroid carcinoma (sMTC) vs. normal controls but so far is not associated with any medical or pathological features of the tumour. The aim of our study was to assess the influence of this variant on the age of onset, clinical, biological and pathological features of sMTC. DESIGN AND PATIENTS: One hundred patients with histologically proven MTC, for whom the germline genetic analysis of RET was negative and medical records were available, were included in the study. RESULTS: Patients with the heterozygous GS variant or the homozygous SS variant (n = 36) were on average 8.0 years younger than patients with the wild-type GG variant (n = 64, mean age 43.9 vs. 51.9 years, P < 0.01). The former group did not differ from the wild-type group in terms of MTC size, prevalence of C-cell hyperplasia (CCH) or papillary thyroid carcinoma (PTC). However, the prevalence of an increased preoperative basal calcitonin (bCT) level (> 1000 pg/ml) was 2.75-fold higher in the patients with the GS or SS variant than in those with the wild-type variant (P < 0.001). The proportion of patients with lymph node metastases was also higher in the former group (P < 0.05). Multivariate analysis confirmed that the presence of the RET variant is independently associated with higher preoperative bCT values (P = 0.011). CONCLUSIONS: Our data demonstrate that the RET G691S variant could modulate the age of onset of sMTC as demonstrated previously for familial tumours. Moreover, this variant is an independent predictor of a higher basal calcitonin synthesis rate in patients with sMTC.

Simultaneous regression of Philadelphia chromosome and multiple nonrecurrent clonal chromosomal abnormalities with imatinib mesylate in a patient autografted 22 years before for chronic myelogenous leukemia.

A 31-year-old patient developed chronic myelogenous leukemia (CML) in November, 1983. In November 1984, following a diagnosis of acceleration, he received an autologous hemopoietic transplant after conditioning with cyclophosphamide and total body irradiation. The autologous marrow was purged with mafosfamide. Over 20 years, the patient remained in chronic phase of CML. Multiple nonrecurrent clonal chromosomal abnormalities appeared leading to a very complex karyotype, including among others involvement of chromosomes 1, 7, 9, 13, 19, and X. Fluorescent in situ hybridization showed that the two chromosomes 9 were involved. Acute myeloid crisis was diagnosed in February, 2004. Treatment with imatinib mesylate resulted within 6 months in a total disappearance of all chromosomal abnormalities with a complete cytogenetic and molecular response, which persists 3 years later. We question whether the ex vivo purging procedure with mafosfamide has favored the occurrence of these particular cytogenetic abnormalities (with no independent oncogenic potential) within the original leukemic stem cell pool. It remains unclear whether the autologous transplantation has indeed resulted into some prolongation of the duration of the chronic phase, which lasted for 20 years. At time of acute crisis, the dramatic response to imatinib mesylate leading to a complete cytogenetic and molecular response is noteworthy.

[A new, rapid and robust genotyping method for CYP2C9 and MDR1]. / Nouvelle méthode rapide et robuste de génotypage du CYP2C9 et MDR1.

Single nucleotide polymorphisms (SNPs) can significantly affect human phenotypes. Detection of allelic variant carriers has become a major goal for clinical pharmacologists in order to study phenotype-genotype relationships. However, there is a crucial need for rapid, and validated pharmacogenetic tests. The aim of the study was to validate a new fluorescence PCR strategy for cytochrome P450 2C9 (CYP2C9) and multidrug resistance gene (MDR1) genotyping. Results of CYP2C9 and MDR1 genotypes determined with reference techniques were compared to those obtained by allelic discrimination assays employing fluorescent TaqMan probes. Sixteen subjects carrying CYP2C9*2 and CYP2C9*3 allelic variants (heterozygous and homozygous) previously identified by sequencing and 55 subjects previously genotyped for MDR1 exon 26 (C3435T) SNP by conventional PCR-RFLP were genotyped with fluorescent PCR. Fluorescent PCR gave 100 % accuracy with the results obtained with reference genotyping strategies for each of the 3 SNPs. Genotyping results with fluorescent PCR repeated on three consecutive occasions remained constant over time for each of the 3 SNPs. Allelic discrimination assays based on fluorescent PCR gave entire satisfaction for CYP2C9 and MDR1 genotyping. This reliable genotyping strategy can be easily used in clinical practice and should be further developed for additional SNPs identification.

Survival and differentiation of porcine hepatocytes encapsulated by semiautomatic device and allotransplanted in large number without immunosuppression.

BACKGROUND/AIMS: The aim of this study was to evaluate the survival and functions of porcine hepatocytes transplanted in large quantities in the peritoneal cavity of allogeneic animals following semiautomatic encapsulation. METHODS: Isolated porcine hepatocytes and a polymer solution composed of AN69 were coextruded through a double lumen spinneret. Minitubes containing hepatocytes were transplanted in the peritoneal cavity of 12 pigs (4 x 10(9) cells/animal) in the absence of immunosuppressive therapy. Seven, 15, and 21 days after transplantation, minitubes was collected and processed for analyses. The morphology was examined under light and electron microscopy. Albumin synthesis was assessed by semi-quantitative reverse transcription-polymerase chain reaction. Cytochrome P450 3A (CYP3A) gene expression was analyzed by Western blot and by testosterone 6-beta-hydroxylation assay. RESULTS: The device allowed to encapsulate 55 x 10(6) hepatocytes/min. Hepatocytes exhibited normal structural and ultrastructural features up to day 21. Albumin gene expression decreased progressively between days 0 and 21. The amount of CYP3A protein and 6-beta-hydroxylase activity were approximately 2-fold lower at days 7 and 15 than in freshly encapsulated hepatocytes, and further decreased thereafter. CONCLUSIONS: The preservation of hepatocyte functions during 1-2 weeks is encouraging for potential short-term use of such bioartificial liver in future clinical application.

Apolipoprotein E polymorphism, a marker of disease severity in primary biliary cirrhosis?

BACKGROUND/AIMS: To determine whether the apolipoprotein E (apo-E) polymorphism is associated with the risk of primary biliary cirrhosis (PBC), the severity of the disease and its response to ursodeoxycholic acid (UDCA) therapy. METHODS: The apo-E genotype was determined in 72 PBC patients. Genotype and allele distributions were compared with those found in the French general population. Laboratory parameters obtained before and after 1- and 4-year UDCA treatment were compared according to the apo-E allele carrier status. RESULTS: Apo-E allele and genotype distributions were similar between PBC patients and the general population. At the time of diagnosis, the epsilon4 allele carriers were younger (P < 0.05), had higher bilirubin (P < 0.05) and IgG (P < 0.001) levels and a lower prothrombin index (P < 0.01) than epsilon2 (homozygous + heterozygous) or epsilon3 homozygous allele carriers. After 4-year UDCA therapy, the decrease in serum alkaline phosphatase and in alanine and aspartate aminotransferase activities was lower in percentage in the epsilon4 than in other epsilon allele carriers (P < 0.01). CONCLUSIONS: Although apo-E polymorphism does not appear to confer susceptibility to PBC, it probably influences PBC progression and response to UDCA. The epsilon4 allele may identify patients with high risk of severe disease and poor response to treatment.

Survival and functions of encapsulated porcine hepatocytes after allotransplantation or xenotransplantation without immunosuppression.

BACKGROUND: This study evaluated the survival and functions of encapsulated porcine hepatocytes after intraperitoneal allotransplantation and xenotransplantation without immunosuppression. METHODS: Isolated porcine hepatocytes were encapsulated in AN 69 polymer capsules (45.10(6)/capsule) and transplanted intraperitoneally in 12 rats and 12 pigs. Fifteen, 30, and 60 days after transplantation, capsules were removed and the viability and morphology of explanted hepatocytes were examined under light and electronic microscopy. The potential to produce albumin was assessed by evaluating the level of albumin messenger RNA, using semiquantitative reverse transcription-polymerase chain reaction. 6beta-Hydroxylase activity was measured by high-performance liquid chromatography. In addition, cytochrome P450 3A proteins were detected by Western blot only in allogeneic hepatocytes. RESULTS: Similar results were observed after allotransplantation and xenotransplantation. Histologic studies showed that hepatocytes were well-preserved and arranged in cords for up to 30 days. The expression of porcine albumin gene was maintained up to 15 days. 6beta-Hydroxylase activity was 2.5-fold lower at day 15 than in freshly encapsulated hepatocytes, which were not transplanted. In allogeneic hepatocytes, the expression of CYP 3A protein was detected up to 60 days after transplantation. CONCLUSIONS: Encapsulated porcine hepatocytes remain viable and functional for at least 15 days after allotransplantation and xenotransplantation without immunosuppression. The demonstration of maintained hepatic functions in transplanted porcine hepatocytes up to 15 days is a first step toward application in the treatment of acute liver failure.

Fetal microchimerism in primary biliary cirrhosis.

BACKGROUND/AIMS: Recent studies have suggested a role of fetal microchimerism in the pathogenesis of scleroderma. The present study investigated the potential role of fetal microchimerism in primary biliary cirrhosis (PBC), a closely related disease. METHODS: A quantitative nested polymerase chain reaction was used to detect Y-chromosome sequences in the peripheral blood or the liver of PBC women and controls having male children and no transfusion or miscarriage history. RESULTS: Male microchimerism was found in the peripheral blood from 45% (9 of 20) of PBC women and 25% (5 of 20) of healthy controls matched for the number of male children and age of the youngest son (p=0.28), and in the liver-biopsy specimens from 33% (5 of 15) of PBC women and 32% (8 of 25) of controls. The level of chimerism did not differ between patients and controls either in blood or in liver. Microchimerism was not related to the severity of the disease but was more frequent in PBC patients with anticentromere antibodies (p=0.049). CONCLUSIONS: Fetal microchimerism does not seem to play a major role in most cases of PBC. However, the association with anticentromere antibodies suggests a possible role in the subgroup of patients with CREST syndrome or scleroderma.

PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury.

The accumulation of myofibroblasts and fibrosis around proliferating bile ducts in cholestatic liver disease has been attributed to the proliferation and phenotypic modulation of portal fibroblasts, whereas the contribution of hepatic stellate cells remains uncertain. There is increasing evidence to indicate that bile ducts may stimulate chemoattraction of hepatic stellate cells (HSC). In the present study, we undertook dynamic tests to examine such a possibility and to investigate the role of two potential mediators: platelet-derived growth factor-BB (PDGF-BB) and endothelin-1. Cholestasis was induced by bile duct ligation in rats. HSC were isolated from normal rats and culture activated into myofibroblasts expressing PDGF-beta receptors. Migration of myofibroblastic HSC was investigated in a Transwell chemotaxis filter assay. As compared with basal conditions, PDGF-BB (100 microg/l) and endothelin-1 (10(-8) M) induced a 3-fold and 1.7-fold increase in HSC migration, respectively. Bile duct segments isolated from cholestatic rats triggered a 3-fold increase in migration. This stimulation was significantly more potent than that observed in the presence of normal bile ducts. It was inhibited by neutralizing anti-PDGF antibodies and by STI571 PDGF receptor tyrosine kinase inhibitor, by 60% and 85%, respectively, whereas Bosentan, an endothelin receptor antagonist, had no significant inhibiting effect. In bile duct segments from cholestatic rats PDGF-B chain mRNA was detected at higher levels than in controls, whereas PDGF-BB was immunolocalized in bile duct epithelial cells. The results indicate that chemotaxis of HSC towards bile duct structures may contribute to the development of periductular fibrosis in cholestatic disorders, and that PDGF-BB is the major mediator in this process. In addition, anti-liver fibrogenic properties of STI571 are suggested by potent inhibition of myofibroblastic HSC function.

[Viability and differentiation of human hepatocytes immunoprotected by macroencapsulation and transplanted in rats]. / Viabilité et état de différenciation des hépatocytes humains immunoprotégés par macroencapsulation et transplantés chez le rat.

OBJECTIVES: To determine the viability and differentiation of human hepatocytes immunoprotected by encapsulation and transplanted in rats without immunosuppression. METHODS: Freshly isolated human hepatocytes were encapsulated in hollow fibers and transplanted in the peritoneal cavity of immunocompetent rats. The fibers were explanted for analysis at D3, D7 and D14 following transplantation. Morphological features under light and electron microscopy and gene expression were compared to those of non-transplanted encapsulated hepatocytes (D0). Human cytochrome P450 3A and albumin mRNAs were quantified by Northern blot. Cytochrome P450 3A proteins were detected by Western blot and cytochrome P450 3A enzyme activity was assessed by measuring the formation of 6beta-hydroxytestosterone by high performance liquid chromatography. RESULTS: Transplanted hepatocytes were more than 60 % viable and exhibited morphological criteria of hepatocytic differentiation up to D7. Albumin and cytochrome P450 3A transcripts were also detected up to D14. At D3 and D7, albumin mRNA levels were of 30 %, compared to control D0 hepatocytes, while cytochrome P450 3A5 and cytochrome P450 3A4 mRNA levels were 65 % and 0 %, respectively. Cytochrome P450 3A immunoreactivity was detected by Western blot up to D14 and 6beta-hydroxylase activity was 17 % at D3 compared to D0, supporting with disappearance of cytochrome P450 3A4 mRNA. CONCLUSIONS: Human hepatocytes remain viable for a short period, following encapsulation and intraperitoneal transplantation in rat. Other experimental conditions need to be tested to prevent or delay a decrease in hepatocyte specific gene expression.

Functional interactions between bile acids, all-trans retinoic acid, and 1,25-dihydroxy-vitamin D3 on monocytic differentiation and myeloblastin gene down-regulation in HL60 and THP-1 human leukemia cells.

Bile acids were shown previously to inhibit proliferation and to induce monocytic differentiation in HL60 human acute promyelocytic leukemia cells (A. Zimber et al., Int. J. Cancer, 59: 71-77, 1994). In this report, we hypothesized that bile acids may exert a positive cooperativity with two known inducers of leukemic cell differentiation, all-trans retinoic acid and 1,25(OH)2-vitamin D3. Our results provide evidence that bile acids induced the monocytic differentiation of HL60 and THP-1 human leukemia cells exposed to ineffective concentrations of these inducers. The protein kinase C (PKC) inhibitors H-7 (10 and 20 microM) and staurosporine (5 and 20 nM) modulated the effects of bile acids on HL60 cell differentiation. Most interestingly, bile acids are shown herein to down-regulate the expression of the serine protease myeloblastin gene involved in the differentiation of myeloid hematopoietic cells. In agreement with the recent identification of nuclear receptors for bile acids, our data suggest that functional interactions between nuclear bile acid signaling pathways, PKC, and nuclear receptors for retinoic acid and vitamin D3 are involved in the down-regulation of the myeloblastin gene and the induction of cell differentiation in human leukemic cells.

Lymphocyte P-glycoprotein expression and activity before and after rifampicin in man.

It has recently been shown that P-glycoprotein (P-gp) is inducible by rifampicin in the human gut as shown in intestinal biopsies. The present study was performed in order to test the hypothesis that human peripheral lymphocytes can be used to assess such an inducibility. We also assessed inter- and intra-individual variability of P-gp expression and activity in peripheral lymphocytes. Blood samples from 13 healthy volunteers were collected 1.7, 14 and 19 days after inclusion. Rifampicin treatment (600 mg/day) was administered from day 15 to day 18. Lymphocyte P-gp expression was measured at the messenger RNA level by semi-quantitative RT-PCR and at the protein level by immunostaining flow cytometry. P-gp activity was determined by flow cytometry with rhodamine 123 efflux. Cytochrome P4503A4 (CYP3A4) inducibility was measured by comparing the urinary metabolic ratio of 6beta-hydroxycortisol/cortisol on day 14 and 19, Lymphocyte P-gp expression and activity was not induced by rifampicin, while it increased CYP3A4 activity from 5.0 +/- 4.0 to 22.9 +/- 16.6 (P < 0.001). There was a 3 - 4-fold inter-individual variability and a 3 - 44 % intra-individual variability of lymphocyte P-gp expression and activity. Peripheral lymphocytes are not an appropriate material to assess P-gp inducibility in humans. P-gp shows significant inter- and intra-individual variability in human lymphocytes.

Low levels of lymphocyte MDR1 gene expression during early renal transplantation in patients treated with tacrolimus.

P-glycoprotein (P-gp) is a membrane efflux pump increasing the transport of drugs such as tacrolimus out of the cells. The aim of the study was to determine the kinetics of lymphocyte P-gp expression in patients treated with tacrolimus during the first 3 months following renal transplantation. Lymphocyte MDR1 gene expression was measured by semi-quantitative RT-PCR a few hours before transplantation, 3 weeks and 3 months after the graft. Lymphocyte MDR1 gene expression was low in all the 10 patients compared to 10 healthy volunteers: 0.30 +/- 0.07 arbitrary units (patients) vs. 1.74 +/- 0. 55 (healthy volunteers) (P = 0.0002). MDR1 gene expression decreased among the patients during the study: 0.28 +/- 0.12 (3 weeks later) and 0.12 +/- 0.09 (3 months later) (P = 0.006). We can conclude that lymphocyte MDR1 gene expression among patients before renal transplantation is low and remains low during the first 3 months following the graft.

Phase I and phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium.

Tissue expression of drug-metabolizing enzymes influences susceptibility to drugs and carcinogens. Because the biliary epithelium, exposed to bile-borne chemicals, may give rise to drug-induced cholangiopathies and to cholangiocarcinomas, we determined the pattern of expression of drug-metabolizing enzymes in this epithelium. We first demonstrated by blot analyses that biliary epithelial cells (BEC) isolated from human gallbladders display cytochrome P450 (CYP) 1A, 2E1, and 3A, microsomal epoxide hydrolase (mEH), alpha, mu, and pi glutathione S-transferase (GST), transcripts and proteins. We also identified CYP-associated steroid 6beta-hydroxylase activity in BEC. CYP and mEH expression was 5- to 20-fold lower in BEC than in autologous hepatocytes, and further differed by a higher ratio of CYP3A5/CYP3A4, and by CYP1A1 predominance over CYP1A2. alphaGST was highly expressed in both hepatocytes and BEC, while piGST was restricted to BEC. In approximately 50% of individuals, muGST was expressed in hepatocytes and at lower levels in BEC. By using the same antibodies as those used in immunoblots, we could show by immunohistochemistry that CYP2E1, CYP3A, mEH, alpha, mu, and piGST immunoreactivities are expressed and display a heterogeneous distribution in the epithelium lining the entire biliary tract except for small intrahepatic bile ducts that were devoid of CYP3A and alphaGST immunoreactivities. In conclusion, BEC contribute to phase II, and although to a lesser extent than hepatocytes, to phase I biotransformation. The distribution of drug-metabolizing enzymes in BEC suggest that they are heterogeneous in their ability to generate and detoxicate reactive metabolites, which may contribute to specific distributions of cholangiopathies.

Endothelin-1 is synthesized and inhibits cyclic adenosine monophosphate- dependent anion secretion by an autocrine/paracrine mechanism in gallbladder epithelial cells.

Ion and fluid transport across the biliary epithelium contributes to bile secretion. Since endothelin (ET)-1 affects ion transport activities and is released by human gallbladder- derived biliary epithelial cells in primary culture, we examined the expression of ET peptides and ET receptors and the influence of ET-1 on ion transport in this epithelium ex vivo. In freshly isolated gallbladder epithelial cells, preproET-1, -2, and -3 mRNAs were detected by reverse transcription PCR and ET-1 isopeptide was identified by chromatography. The cells also displayed ET receptor mRNAs and high-affinity binding sites for ET-1, mostly of the ETB type. Electrogenic anion secretion across intact gallbladder mucosa was stimulated by forskolin, secretin, and exogenous ATP, as assessed by short-circuit current (Isc) increases in Ussing-type chambers. ET-1 inhibited forskolin- and secretin-induced changes in Isc, without affecting baseline Isc or ATP-induced changes. Accordingly, ET-1 significantly reduced the accumulation of intracellular cAMP elicited by forskolin and secretin in the epithelial cells, and this effect was abolished by pertussis toxin. This is the first evidence that ET-1 is synthesized and inhibits, via a Gi protein-coupled receptor, cAMP-dependent anion secretion in human gallbladder epithelium, indicating a role in the control of bile secretion by an autocrine/paracrine mechanism.

Circulating albumin messenger RNA in hepatocellular carcinoma: results of a multicenter prospective study.

The presence of circulating tumor cells might be an indicator of hematogenous spread of tumor cells leading to extrahepatic metastasis. Messenger RNA (mRNA) expression of human albumin, as a liver specific cell marker, has been proposed for this purpose in hepatocellular carcinoma. We conducted a multicenter prospective study in 101 patients with biopsy-proven hepatocellular carcinoma followed-up every 3 months for 1 year or until death. At entry into the study, albumin mRNA was detected in the blood by reverse transcription-polymerase chain reaction (RT-PCR). At entry into the study, 45% of the patients had a positive albumin mRNA test, 53% a single tumor, 16% a portal or venous hepatic thrombosis, and 16% had proven metastasis. After 1 year, there was no significant difference in survival of patients with positive or negative albumin mRNA at entry (P = .16, log-rank test). When patients with metastasis at entry were excluded, again survival did not differ between the two groups (P = .20). Independent prognostic factors of survival were radical therapeutic procedures, metastasis, number of tumors, Child-Pugh score, and thrombosis, but not the albumin mRNA test. Taking the presence of metastasis as a reference, the specificity of the test was 56%, its sensitivity 50%, and its negative predictive value 85%. The present study shows that circulating albumin mRNA detected by means of RT-PCR fails to provide significant information in the diagnosis and prognosis of hepatocellular carcinoma. Further studies are needed to determine whether the use of specific tumor markers could have clinical relevance in this setting.

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.