The dual mTORC1 and mTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.

[Complement factor I deficiency revealed by repeated systemic Streptococcus pneumoniae infection]. / Déficit homozygote en facteur I du complément révélé par une infection systémique répétée à Streptococcus pneumoniae.

Recurrent and serious otitis media, and 2 Streptococcus pneumoniae bacteraemia episodes evoked an immune system deficiency in a 6-year-old girl. Upon investigation of the complement system, CH50 activity was moderately reduced and C4 antigen level was normal contrasting with low C3 antigen level. Factor 1 was undetectable. Factor I deficiency is rare, and its diagnosis has important practical consequences. Thanks to preventive antibiotic therapy with penicillin V and vaccinations against Neisseria meningitidis and S. pneumoniae, life expectancy and quality of this child can be significantly improved.

Identification and molecular characterization of CALM/AF10fusion products in T cell acute lymphoblastic leukemia and acute myeloid leukemia.

The t(10;11)(p12-p13;q14-q21) observed in a subset of patients with either acute lymphoblastic leukemia or acute myeloid leukemia has been shown to result in the fusion of AF10 on chromosome 10 with CALM (also named CLTH) on chromosome 11. AF10 was originally identified as a fusion partner of MLL in the t(10;11)(p12-p13;q23) observed in myeloid leukemia. CALM is a newly isolated gene, cloned as the fusion partner of AF10 in the monocytoid cell line, U937. In order to understand the relationship between MLL, AF10, CALM and the leukemic process, fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction were used to study a series of nine leukemia patients with a t(10;11). Six had myeloid leukemia (AML-M0, AML-M1, AML-M4 and AML-M5) and three had T cell lymphoblastic leukemia. We identified four different CALM/AF10 fusion products in five patients and AF10/CALM reciprocal message in one. We conclude that fusion of CALM and AF10 is a recurring abnormality in both lymphoid and myeloid leukemias of various types including AML-M5, and that the breakpoints in the two types of leukemia do not differ. Our data indicate that the CALM/AF10 fusion product on the der(10) chromosome is critical to leukemogenesis. Leukemia (2000) 14, 100-104.

Establishment and characterization of a megakaryoblast cell line with amplification of MLL.

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.

Results of surgical management of internal carotid artery aneurysm by the cervical approach.

From 1981 to 1995 a total of 14 patients with a mean age of 52 years (range: 23-71) underwent surgery for 15 aneurysms of the extracranial internal carotid artery. Fusiform aneurysms of the carotid bifurcation were not included in this study. Aneurysm led to brain ischemia in 10 cases and rupture in one case. In the remaining four cases, aneurysm was asymptomatic including three that were detected following hemispheric stroke related to a contralateral aneurysm. The etiology was spontaneous dissection in four cases, blunt trauma in three cases, fibromuscular disease in five cases, and atheroma in three cases. The upper limit of the aneurysm was located at C1-C2 in six cases, at C1 in three cases, and above C1 (at the base of the skull) in six cases. The cervical approach was used to successfully perform 12 revascularizations and three ligations (including one after extra-intracranial bypass). There were no postoperative deaths. One transient ischemic attack (TIA) occurred after ligation. Peripheral facial paralysis (PFP) occurred in four of the nine cases in which an extended cervical approach was used. No patients were lost to follow-up. Mean duration of follow-up was 4 years (range: 2 months-10 years). Two patients died at 2 and 4 years of causes unrelated to the procedure. All carotid reconstructions are currently patent and no neurologic manifestations have occurred. PFP persisted in one case. The results of this series confirm that surgical therapy of aneurysms of the extracranial internal carotid artery achieves satisfactory short- and medium-term results and that the extended cervical approach allows treatment of lesions near the base of the skull.

Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping.

Cytogenetic analysis provides critical information of diagnostic and prognostic importance for haematological malignancies. In fact, the identification of recurring chromosomal breakpoints in leukaemias and lymphomas has expedited the cloning of genes whose translocation-induced deregulation causes malignant transformation. The pillar of karyotype analysis rests on chromosome banding techniques that have the distinct advantage that the entire genome can be analysed in a single experiment. However, poorly spread or contracted metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, common in tumour cell preparations, are often difficult to interpret unambiguously and subtle chromosomal aberrations, in particular the exchange of telomeric chromatin or small insertions remain elusive. Fluorescence in situ hybridization (FISH) overcomes some of these limitations, but is mainly utilized to confirm the presence of previously characterized or suspected aberrations. We have developed a novel approach, termed spectral karyotyping or SKY based on the hybridization of 24 fluorescently labelled chromosome painting probes that allows the simultaneous and differential colour display of all human chromosomes. We have used SKY to complement conventional banding techniques in haematological malignancies by analysing 15 cases with unidentified chromosome aberrations. In all instances SKY provided additional cytogenetic information, including the identification of marker chromosomes, the detection of subtle chromosomal translocations and the clarification of complex chromosomal rearrangements. Thus, SKY in combination with standard chromosome banding allows the characterization of chromosomal aberrations in leukaemia with unprecedented accuracy.

AF10 is split by MLL and HEAB, a human homolog to a putative Caenorhabditis elegans ATP/GTP-binding protein in an invins(10;11)(p12;q23q12).

Invins(10;11)(p12;q23q12) is one of the rare but recurring chromosome rearrangements seen in acute monoblastic leukemia. We cloned the proximal 10p breakpoint from one patient and showed that the MLL gene at 11q23 was fused to the 3' portion of AF10 at 10p12. In addition, we cloned the telomeric 10p junction and we found that the 5' portion of AF10 was juxtaposed to a previously unidentified gene at 11q12, which we call HEAB (a human homolog to a hypothetical Caenorhabditis elegans ATP/GTP-binding protein). These results indicate that the AF10 gene is split into a 5' AF10 and a 3' AF10 portion by the 11q23q12 chromosome segment and that both breakpoint junctions result in fusion transcripts of 5' AF10/HEAB and MLL/3' AF10. Only the MLL/3' AF10 fusion mRNA results in an in-frame fusion. Northern blot analysis of HEAB expression shows that a 2.0-kb major transcript is expressed ubiquitously in human tissues and is especially abundant in testis and skeletal muscle, whereas a 3.2-kb minor transcript is noted with the highest level of expression in thymus and peripheral blood leukocytes. The HEAB gene encodes a 425-amino acid protein that is rich in valine and leucine. HEAB protein shows high homology in its entire amino acid sequence to a putative C elegans protein and contains an adenosine triphosphate (ATP)/guanosine triphosphate (GTP)-binding motif that has homology to the ATP-binding transporter superfamily or to GTP-binding proteins. Our results could explain the high frequency of complex insertion and other rearrangement events that involve 10p12 and 11q12 and 11q23. The finding that different portions of a single gene are involved in fusions with two independent genes in the same leukemic cell is unique in the analysis of chromosome translocations.

Correlation between the ETV6/CBFA2 (TEL/AML1) fusion gene and karyotypic abnormalities in children with B-cell precursor acute lymphoblastic leukemia.

The recently identified ETV6/CBFA2 (formerly known as TEL/AML1) fusion gene occurs as a result of the t(12;21)(p12;q22). Initial reports have indicated that the fusion transcript occurs in up to 30% of children diagnosed with B-cell precursor (CD10+, CD19+) acute lymphoblastic leukemia (ALL). In order to characterize the incidence of the t(12;21) at both the chromosomal level as well as the RNA transcript level, we have used a combination of classical cytogenetics, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) to examine the bone marrow of 34 children diagnosed with B-cell precursor ALL. Nine of the 34 patient samples expressed the ETV6/CBFA2 transcript. When the results of RT-PCR were compared with the conventional karyotype, the fusion was present in 3 of 10 (33%) with chromosome 12 abnormalities, none of whom had an obvious t(12;21). The transcript was also detected in 5 of the 12 (41%) bone marrow samples with other abnormalities and in 1 of 12 (8%) samples with a normal karyotype. Seven of the 9 RT-PCR positive patient samples were studied with FISH. Of the 7, FISH confirmed the ETV6/CBFA2 fusion in 6. One other patient with a 12p abnormality had evidence for the fusion using FISH which was not detected by RT-PCR. Our results not only confirm that the frequency of the t(12;21) is unusually high in childhood B-cell precursor ALL, but also that none of the translocations in our series was detected with conventional cytogenetic techniques.

TEL-AML1 translocations with TEL and CDKN2 inactivation in acute lymphoblastic leukemia cell lines.

The t(12;21) (p 13; q22) results in the fusion of the TEL gene located on chromosome 12 with the AML1 gene located on the derivative chromosome 21. Because this translocation is difficult to detect using standard cytogenetic techniques, 27 previously karyotyped B-lineage acute lymphoblastic leukemia (ALL) cell lines were evaluated for the presence of the TEL-AML1 fusion using the reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and cDNA sequencing. Six cell lines expressed the TEL-AML1 chimeric transcript by RT-PCR and the t(12;21) was confirmed by FISH analysis with probes for TEL, AML1, and chromosome 12. While only one of the 6 cell lines with the t(12;21) lost the der(12)t(12;21)-encoded AML1-TEL fusion transcript, 4 cell lines lacked expression of the nontranslocated allele of TEL and 5 cell lines lacked expression of CDKN2. Moreover, in 2 patients (1 with the TEL-AML1 transcript and 1 without), TEL expression was lost with disease progression; le, TEL was expressed in the initial cell lines (established at diagnosis or first relapse) whereas TEL was not expressed in the cell lines established from these patients in late-stage disease. These data show the coexistence of multiple genetic defects in childhood B-lineage ALL Cell lines with t(12;21) will facilitate the study of TEL-AML1 and AML1-TEL fusion proteins as well as TEL and CDKN2 gene inactivation in leukemia transformation and progression.

Analysis of the t(6;11)(q27;q23) in leukemia shows a consistent breakpoint in AF6 in three patients and in the ML-2 cell line.

The t(6;11)(q27;23) is one of the most common translocations observed in patients with acute myeloid leukemia (AML). The translocation breakpoint involves the MLL gene, which is the human homolog of the Drosophila trithorax gene, at 11q23 and the AF6 gene at 6q27. Reverse transcriptase-polymerase chain reaction (RT-PCR) using an MLL sense primer and an AF6 antisense primer detected the MLL/AF6 fusion cDNA from three leukemia patients with the t(6;11) [two AML and one T-acute lymphoblastic leukemia (ALL)] and one cell line. The fusion point in the AF6 cDNA from these cases is identical, regardless of the leukemia phenotype. The ML-2 cell line, which was established from a patient with AML that developed after complete remission of T-cell lymphoma, has retained an 11q23-24 deletion from the lymphoma stage and has acquired the t(6;11) with development of AML. The ML-2 cells have no normal MLL gene on Southern blot analysis, which indicates that an intact MLL gene is not necessary for survival of leukemic cells.

[Echo-Doppler for early diagnosis of deep venous thrombosis in orthopedic surgery and traumatology. A retrospective study of 1,647 patients]. / Echo-Doppler pour le diagnostic précoce des thromboses veineuses profondes en chirurgie orthopédique et traumatologique. A propos de 1,647 malades explorés.

UNLABELLED: The authors report the results of duplex ultrasound scanning investigation for the detection of deep venous thrombosis after orthopedic surgery and compare asymptomatic and symptomatic populations. PURPOSE OF THE STUDY: To estimate the rate of deep venous thrombosis diagnosed by duplex scanning in those 2 populations and precise their features. MATERIAL: A total of 1,647 in-patients all receiving low-molecular-weight heparin and investigated from 1989 to 1993. either for screening because of high risk of thrombosis (asymptomatic group: 930 patients, mean age +/- SD 63 +/- 17 years) or for clinical suspicion of deep vein thrombosis (symptomatic group; 717 patients, mean age +/- SD: 57 +/- 21 years). Difference between the two groups mean ages were significant (p < 10(-8). METHODS: An Hitachi EUB 450 duplex and an Acuson colour duplex 128 XP, with 3.5 MHz and 7.5 MHz linear probes were used. Veins were tested for compressibility in transverse view from caval site to both ankles. Retrospective analysis of patients database results has been achieved. RESULTS: There was no significant difference in deep vein thrombosis rate between screening asymptomatic group (356/930:38%) and symptomatic group (253/717: 35%). There was a linear relation, in the 2 groups, between age and deep vein thrombosis rate, from 10% before twenty to 45% after eighty years old. For a relative risk to have thrombosis detected before twenty definite at 1, it was 2.1 for 20-29, 4.9 for 40-49, 6.2 for 60-69 and 8.6 later than 80 years old. Proximal deep vein thrombosis was detected in only 5% (87/1,647) of patients. Distal muscular soleal veins were the most usual involved sites of thrombosis. Isolated soleal thrombosis were detected in 16% (270/1,647) of patients. There was non significant difference between the deep vein thrombosis rate after total knee or hip arthroplasty among selected patients for duplex scanning from 1989, and the true prevalence assessed among all the patients who have undergone total hip or knee arthroplaty during the last 6 months. DISCUSSION: Pessimistic results previously reported for duplex screening among asymptomatic patients are not confirmed. Calf vein thrombosis rate assessed by duplex scanning exceeds by 15 a 20% usual rates assessed by contrast venography, among patients receiving low-molecular-weight heparins. That difference could be assigned to the isolated muscular soleal thrombosis usually missed at contrast venography. CONCLUSION: Deep vein thrombosis rate among orthopedic surgical patients, is much higher when detected with Duplex ultrasound scanning than detected with contrast venography, and is related to patient age. Screening for deep venous thrombosis by duplex scanning in orthopedic surgery is as efficient among asymptomatic as among symptomatic patients and could become soon a systematic screening. Soleal vein thrombosis are the most usual. Mechanical calf venous pump stimulation in association with low molecular weight heparin, has to be evaluated in attempting to reduce those muscular soleal veins thrombosis.

[Detection of deep vein thrombosis of the limbs by ultrasound in orthopedic and traumatologic surgery. Results of 1647 studied patients]. / Dépistage des thromboses veineuses profondes des membres inférieurs par écho-doppler en chirurgie orthopédique et traumatologique. Résultats sur 1647 malades explorés.

INTRODUCTION: This study was undertaken to estimate the efficiency of duplex ultrasound scanning and its utility to detect deep vein thrombosis in orthopedic patients and to describe their features. MATERIAL AND METHODS: A total of 1647 in-patients, all receiving low-molecular-weight heparin, were investigated from 1989 to 1993, either for screening because of a high risk of thrombosis (asymptomatic group: 930 patients, mean age + SD: 63 + 17 years) or for clinical suspicion of deep vein thrombosis (symptomatic group: 717 patients, mean age + SD: 57 + 21 years). Difference between the two groups mean ages was significant (p < 10(-8)). An Hitachi EUB 450 duplex and an Acuson colour duplex 128 XP, with 3.5 MHz and 7.5 MHz linear probes were used. Veins were tested for compressibility in the transverse view from caval site to both ankles. Retrospective analysis of patients' database results was done. RESULTS: There was no significant difference in deep vein thrombosis rate between screening asymptomatic group (356/930: 38 per cent) and symptomatic group (2531717: 35 per cent). There was a linear relation, in the 2 groups, between age and deep vein thrombosis rate, from 10 per cent before twenty to 45 per cent after eighty years old. For a relative risk to have thrombosis detected before twenty definite at 1, it was 2.1 for 20-29, 4.9 for 40-49, 6.2 for 60-69 and 8.6 later than 80 years old. Proximal deep vein thrombosis was detected in only 5 per cent (87/1647) of patients. Distal muscular soleal veins were the most usual involved sites of thrombosis. Isolated soleal thrombosis were detected in 16 per cent (270/1647) of patients. There was no significant difference between the deep vein thrombosis rate after total knee or hip arthroplasty among selected patients for duplex scanning from 1989, and the true prevalence assessed among all the patients who have undergone total hip or knee arthroplasty during the last 6 months. DISCUSSION: Pessimistic results previously reported for duplex screening among asymptomatic patients are not confirmed. Calf vein thrombosis rate assessed with duplex exceeds by 15 to 20 per cent the rates assessed by contrast venography, among patients receiving low molecular weight heparins. That difference could be attributed to the isolated muscular soleal thrombosis usually missed at contrast venography. CONCLUSION: Deep vein thrombosis rate among orthopedic surgical patients, is much higher when detected with Duplex ultrasound scanning than detected with contrast venography, and is related to patient age. Soleal vein thrombosis is the most prevalent. Duplex ultrasound scanning is an efficient and useful screening method for deep vein thrombosis in orthopedic surgery. Mechanical calf venous pump stimulation in association with low molecular weight heparin, has to be evaluated in attempting to reduce those muscular soleal veins thrombosis.

[Sudden death in patients wearing pacemakers]. / La mort subite chez le porteur de stimulateur cardiaque.

10 to 30 p. cent of patients with cardiac pacemakers die suddenly. In most cases, the cause of death can not be established. Racing of the pacemaker is a rare but definite cause of sudden death. In contrast, failure of the stimulation, which occurs much more frequently, and which is due to failure of the pacemaker or the wire or to an elevation of the threshold of stimulation, rarely causes sudden death because of the development of an idioventricular rhythm, leading to detection of the fault. Competitive rhythms do not appear to be more dangerous than accidental inhibition in sentinel pacemakers; both of these mechanisms can lead to ventricular tachycardia which may degenerate to ventricular fibrillation. Programmable pacemakers have certain advantages and disadvantages, in that the programming may prove to be inappropriate. In most cases, the ventricular fibrillation is spontaneous, occurring in the context of myocardial failure. The extension of the indications for pacemakers is certainly responsible for the relatively high incidence of sudden death.

Further histochemical studies on masticatory muscles.

This paper describes a histochemical and histographic analysis of the masticatory muscles obtained from 78 early autopsy samples from subjects from 4 days to 87 years old. Five groups of muscles have been stuied: the temporalis, the medial and lateral pterygoid, the superficial bundle of the masseter and the mylohyoideus. All adult muscles have consistently shown a markedly increased number oftype II fibres and a disparity in the size of the two main fibre types, the average diameter of type II fibres being about half that of type I fibres. Fibres of intermediate size and stain were observed with myofibrillar ATPase at pH 9.40. A negative relation between the percentage of type II fibres and intermediate fibres was found, but not between the percentage of type I fibres and of intermediate fibres. Another negative relation was found between the number and the size of type II fibres, again not present in type I nor in intermediate fibres. In children, from 6 days old, an increased number of type II fibres and a definite disparity in the size of the two main fibre types were found. Intermediate fibres were present on the 17th day. Up to the age of 13 years, their diameter was greater than that of type I fibres. The analysis of the distribution and size modifications of the various fibre types seems to indicate a progressive adaptation of the masticatory muscles. This adaptation of the fibres to the successive reactions and to the various movements of the masticatory system is then discussed.

Effects of disodium dichloromethylene diphosphonate on Paget's disease of bone.

19 patients with Paget's disease were treated orally for 6 months with disodium dichloromethylene diphosphonate. 1600 mg/day (10 patients) significantly reduced urine hydroxyproline, serum alkaline phosphatase, urine calcium, and the number of pagetic bone osteoclasts. Tetracycline double labelling revealed undisturbed bone mineralisation. There was improvement on quantitative bone-scans and bone pain diminished. There was a transient increase in parathyroid hormone level in 13 of the 19 patients during treatment, which was associated with a high serum 1,25 (OH)2D3. No adverse clinical side-effects have been observed and biochemical remission has persisted for 9 months.

[Oral nerve endings in the cat]. / Terminaisons nerveuses buccales chez le chat.

The oral nerve endings of the palate, the buccal mucosa and the periodontal ligament of the cat canine were characterized by the presence of a cellular envelope which is the final form of the Henle sheath. The structures described as end-rings in the periodontal ligament are more probably subterminal nervous structures rather then receptors. They are not specific to the ligament. They can be observed in the palate and in the buccal mucosa where, as in the periodontal ligament, mechano-receptors of the Vater-Pacini type can be seen.

The histochemical profile of the human masseter. An autopsy and biopsy study.

A histochemical study has been carried out upon samples of muscle obtained from the human masseter. Sixteen specimens obtained either at autopsy or biopsy have shown that the middle and deep portions of the muscle contain fibres which in their size, histochemical staining properties and number correspond to the appearances noted in most human limb skeletal muscles. By contrast, samples taken from the superficial portion of the masseter demonstrated several unusual characteristics: these included a striking inequality of muscle fibre size in that the Type II fibres were much smaller than those of Type I but exceeded the latter in total number. This part of the muscle also contained, in 6 out of 10 samples, substantial numbers of intermediate fibres identified by myofibrillar ATPase staining. This study has confirmed that the superficial part of the human masseter is different morphologically and presumably functionally from the middle and deep parts of the muscle.