Orbital decompression surgery for thyroid eye disease: implications for anaesthesia.

The management of thyroid-associated eye disease is reviewed with particular reference to surgical management and its implications for anaesthetists. Experience from a unit undertaking such surgery is presented.

In vitro drug-resistance profile in infant acute lymphoblastic leukemia in relation to age, MLL rearrangements and immunophenotype.

Acute lymphoblastic leukemia (ALL) in infants under 1 year is strongly associated with translocations involving 11q23 (MLL gene), CD10-negative B-lineage (proB) immunophenotype, and poor outcome. The present study analyses the relationship between age, MLL rearrangements, proB-lineage, and in vitro drug resistance determined using the MTT assay. Compared to 425 children aged over 1 year with common/preB (c/preB) ALL, the 44 infants were highly resistant to steroids (for prednisolone (PRED) more than 580-fold, P=0.001) and L-asparaginase (L-ASP) (12-fold, P=0.001), but more sensitive to cytarabine (AraC) (1.9-fold, P=0.001) and 2-chlorodeoxyadenosine (2-CdA) (1.7-fold, P<0.001). No differences were found for vincristine, anthracyclines, thiopurines, epipodophyllotoxines, or 4-hydroperoxy (HOO)-ifosfamide. ProB ALL of all ages had a profile similar to infant ALL when compared with the group of c/preB ALL: relatively more resistant to L-ASP and PRED (and in addition thiopurines), and more sensitive to AraC and 2-CdA. Age was not related to cellular drug resistance within the proB ALL group (<1 year, n=32, vs >/=1 year, n=19), nor within the MLL-rearranged ALL (<1 year, n=34, vs >/=1 year, n=8). The translocation t(4;11)(q21;q23)-positive ALL cases were more resistant to PRED (>7.4-fold, P=0.033) and 4-HOO-ifosfamide (4.4-fold, P=0.006) than those with other 11q23 abnormalities. The expression of P-glycoprotein, multidrug-resistance protein, and lung-resistance protein (LRP) was not higher in infants compared to older c/preB ALL patients, but LRP was higher in proB ALL and MLL-rearranged ALL of all ages. In conclusion, infants with ALL appear to have a distinct in vitro resistance profile with the proB immunophenotype being of importance. The role of MLL cannot be excluded, with the t(4;11) being of special significance, while age appears to play a smaller role.

In vitro drug resistance profile of Philadelphia positive acute lymphoblastic leukemia is heterogeneous and related to age: a report of the Dutch and German Leukemia Study Groups.

BACKGROUND: The t(9;22)(q34;q11) translocation leading to the Philadelphia (Ph) chromosome resulting in BCR-ABL gene fusion is associated with a poor prognosis in acute lymphoblastic leukemia (ALL). PROCEDURE: We studied the relation between t(9;22), determined by karyotype, fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR), and in vitro drug resistance, measured by the MTT assay, in precursor B-cell ALL at diagnosis. The findings in twenty-one Ph-positive (Ph+) childhood common/precursorB (c/preB) cases were compared with 254 Ph-negative (Ph-) ALL cases. RESULTS: A large range of LC(50) values was found within the Ph+ patients. Moreover, LC(50) values did not differ significantly between Ph+ and Ph- samples for prednisolone, dexamethasone, L-asparaginase, vincristine, anthracyclines, thiopurines, epipodophyllotoxins, and 4H00-ifosfamide, even after matching for important prognostic features (age, white blood cell count (WBC), and immunophenotype). Adult Ph+ (n = 12) ALL was more resistant to prednisolone (> 270-fold, P = 0.030), and displayed an overall tendency to resistance when compared to matched cases of Ph- (n = 15) adult precursor B-cell ALL. Within Ph+ ALL, in vitro prednisolone resistance increased significantly with age (P = 0.006). The expression of lung resistance protein (LRP), but not P-glycoprotein (P-gp) or multidrug resistance protein (MRP), was significantly higher in all Ph+ patients. CONCLUSIONS: Both childhood and adult Ph+ precursor B-cell ALL samples display a heterogeneous in vitro resistance profile, with relatively sensitive and resistant cases. The adult Ph+ samples, however, are generally more resistant compared to matched Ph- controls, reaching significance for prednisolone. The correlation of prednisolone resistance with age within the Ph+ cases might help explain the poorer prognosis of adult Ph+ ALL.

t(7;12)(q36;p13) and t(7;12)(q32;p13)--translocations involving ETV6 in children 18 months of age or younger with myeloid disorders.

Our retrospective karyotype review revealed two rare recurrent translocations affecting ETV6 (TEL): t(7;12)(q36;p13) and t(7;12)(q32;p13). Five patients with a t(7;12) were from a group of 125 successfully karyotyped pediatric patients enrolled in consecutive clinical AML trials of the Dutch Childhood Leukemia Study Group over a period of 7 years. During a search of available cytogenetic databases, we found 7q and 12p abnormalities in two additional Dutch patients and in three participants in Pediatric Oncology Group trials. A del(12p) had been initially identified in four of these patients and re-examination of the original karyograms revealed a t(7;12)(q36;p13) in two instances and a probable t(7;12) in the other two. FISH confirmed the presence of a t(7;12)(q36;p13) in the latter. Most (n = 7) also had trisomy 19. The t(7;12)(q36;p13) (n = 9) was more common than the t(7;12)(q32;p13) (n = 1). These subtle translocations were found only in children 18 months of age or younger. A literature search revealed that the t(7;12) with break-points at 7q31-q36 and 12p12-p13 had been reported in six children with myeloid disorders and in two with acute lymphoblastic leukemia; all were 12 months of age or younger. Only two of the 17 for whom survival data were available, were alive after at least 22 months of continuous complete remission. Our findings suggest that ETV6 rearrangements due to a t(7;12) may play an adverse role in myeloid disorders in children 18 months of age or younger. Therefore, children in this age group with myeloid disorders should be screened for both MLL and ETV6 rearrangements.

In vitro drug resistance and prognostic impact of p16INK4A/P15INK4B deletions in childhood T-cell acute lymphoblastic leukaemia.

p16 gene deletions are present in about 70% of primary paediatric T-cell acute lymphoblastic leukaemia (T-ALL) and 20% of common/precursor B-cell ALL cases. It is not clear what the impact of the frequent p16 deletions is within the subgroup of T-lineage ALL. We studied the relationship between p16/p19ARF deletions, using fluorescence in situ hybridization, and in vitro drug resistance and prognosis in childhood T-ALL at diagnosis. The cellular drug resistance was measured with the methyl thiazol tetrazoliumbromide assay using a panel of drugs and the thymidylate synthase inhibition assay for methotrexate. There was a complete overlap of individual LC50 values of p16 gene homozygously deleted and p16 germ-line cases for most of the nine classes of drugs tested. The only difference was for dexamethasone: the p16-deleted group was more sensitive than the germ-line p16 group (P = 0.030). The homozygously deleted p16 T-ALL patients (n = 34) treated with the modern multiagent chemotherapy schemes of the Dutch Childhood Leukaemia Study Group ALL-VII/-VIII or Co-operative ALL-92/-97 protocols have a significantly lower 5-year disease-free survival (DFS) than germ-line p16 T-ALL (n = 25) (65.1 +/- 9.1% vs. 95.5 +/- 4.4%, Plog rank = 0.021). Hence, this study identifies a subpopulation of primary childhood T-ALL that appears to have an extremely high DFS. However, the observed differences in outcome do not seem to be related to intrinsic resistance for the tested drugs.

MDR1 expression in poor-risk acute myeloid leukemia with partial or complete monosomy 7.

Expression of the multidrug resistance (MDR1) phenotype, encoded by the MDR1 gene, is an adverse prognostic factor for CR and survival in acute myeloid leukemia (AML). Other prognostic factors, such as specific cytogenetic abnormalities, have been identified in AML. We have investigated the expression of the MDR1 gene in untreated AML patients with monosomy 7 (n = 12), and partial deletions (n = 7) of the long arm of chromosome 7 (respectively -7/7q-), because of the extremely bad prognosis associated with these cytogenetic abnormalities and because of the fact that the MDR1 gene is located on chromosome 7q21.1. The findings were compared with the level of MDR1 expression in a group of 42 other AML patients, matched for age with favourable, neutral or complex cytogenetic abberations. MDR1 mRNA expression, as measured by the RNase protection assay was significantly higher in the -7/7q- group vs other AML patients (median 1.3 vs 0.1 arbitrary units, P = 0.02). Protein expression of MDR1 in the -7/7q- group, as determined with the monoclonal antibody MRK16, was found to be similar to the levels found in the control group. With a functional rhodamine retention assay using the modulator PSC833, increased MDR1 activity was observed in the -7/7q- group as compared to the control group of patients (P = 0.05). Considering the higher MDR1 mRNA expression and equal or slightly elevated level of protein expression of MDR1, we studied the presence of MDR1 genes in this group of -7/7q- patients. Fluorescence in situ hybridization (FISH) studies, using a specific MDR1 probe revealed no loss of an MDR1 allele in any of the deleted q- arms of the seven patients with 7q-, whereas all monosomy 7 patients lacked one MDR1 gene homologue. To determine whether there was selective loss of the MDR1 gene in the -7/7q- patients, the genetic polymorphism of the MDR1 gene was used. Both allelic variants (G and T) were represented in the -7/7q- and in the control group, showing a predominance for GT at position 2677 of the MDR1 gene in the control group. In the 12 monosomy 7 patients loss of the MDR1 allele was random. Methylation studies of the CpG island of the MDR1 gene revealed no hypermethylation in any of the -7/7q- patients. We conclude that MDR1 expression in -7/7q- AML patients is upregulated at transcriptional, but not at translational level, suggesting that mechanisms other than MDR1 are responsible for the poor prognosis in these patients.

Characterization of complex chromosomal abnormalities in uveal melanoma by fluorescence in situ hybridization, spectral karyotyping, and comparative genomic hybridization.

Several nonrandom recurrent chromosomal changes are observed in uveal melanoma. Some of these abnormalities, e.g., loss of chromosome 3, gain of the q arm of chromosome 8, and chromosome 6 abnormalities, are of prognostic value. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) are used to detect these changes. In some cases, however, detailed cytogenetic analysis is not possible due to the presence of complex abnormalities. To define more accurately these cytogenetic changes, we have applied comparative genomic hybridization (CGH) and/or spectral karyotyping (SKY) to two uveal melanoma cell lines and five primary uveal melanomas, with partially defined and/or complex abnormalities. SKY provided additional information on 34/39 partially defined aberrant chromosomes and revealed a new abnormality, a der(17)t(7;17)(?;q?), that had not been recognized by conventional cytogenetics. Additionally, using SKY, abnormalities involving chromosome 6 or 8 were found to be twice as common as observed with cytogenetic analysis. CGH was especially useful in assigning the abnormalities identified by SKY to specific chromosomal regions and, in addition, resulted in the detection of a small deletion of chromosome region 3q13 approximately 21. We conclude that SKY and CGH, as methods complementary to cytogenetic and FISH analysis, provide more complete information on the chromosomal abnormalities occurring in uveal melanoma.

TEL/AML1 gene fusion is related to in vitro drug sensitivity for L-asparaginase in childhood acute lymphoblastic leukemia.

The t(12;21) translocation resulting in TEL/AML1 gene fusion is present in approximately 25% of patients with precursor B-lineage pediatric acute lymphoblastic leukemia (ALL). Studies suggest an association with a good prognosis; however, relapse can occur. We studied the relation between t(12;21), determined by fluorescence in situ hybridization or polymerase chain reaction, and in vitro drug resistance, measured by the MTT assay, in childhood B-lineage ALL at diagnosis. A total of 180 ALL samples were tested, 51 (28%) of which were positive for t(12;21). The median LC(50) values did not differ significantly between TEL/AML1-positive and -negative samples for prednisolone, dexamethasone, daunorubicin, thiopurines, epipodophyllotoxins, and 4-HOO-ifosfamide. However, the TEL/AML1-positive patients were relatively more sensitive to L-asparaginase (ASP; 5.9-fold; P =.029) and slightly but significantly more resistant to vincristine (1.5-fold; P =.011) and cytarabine (1.5-fold; P =.014). After matching for unevenly distributed patient characteristics-that is, excluding patients younger than 12 months, patients with CD10-negative immature B-lineage ALL, patients with Philadelphia chromosome, and patients who were hyperdiploid (more than 50 chromosomes) from the TEL/AML1 negative group-the only remaining difference was a relative sensitivity for ASP in the TEL/AML1-positive samples (10.8-fold; P =. 012). In conclusion, the presence of TEL/AML1 gene fusion in childhood precursor B-lineage ALL does not seem to be associated with a high in vitro drug sensitivity, except for ASP, indicating that these patients could benefit from treatment schedules with significant use of this drug.

Cytogenetic clonality analysis of megakaryocytes in myelodysplastic syndrome by dual-color fluorescence in situ hybridization and confocal laser scanning microscopy.

In the myelodysplastic syndrome (MDS), cytogenetic abnormalities are often present and can be used as markers in studies for cell lineage involvement. Little is known of the involvement of the megakaryocytic lineage due to the variable ploidy of these cells. We applied dual-color fluorescence in situ hybridization (FISH) to routinely prepared bone marrow (BM) smears of cytogenetically normal patients and seven patients with MDS and monosomy 7 or trisomy 8. Probes specific for the centromeric regions of chromosomes 7 and 8 were detected with fluorescein isothiocyanate (FITC) and Texas Red, respectively. This enabled us to assess the ratio between the numbers of chromosomes 7 and 8 in the polyploid cells. We utilized confocal laser scanning microscopy to count the FITC and Texas Red FISH signals in the different focal layers of the megakaryocytes. Fifty-six megakaryocytes in six normal BM smears were analyzed, giving a mean ratio of 1.0, a standard deviation (SD) of 0.12, and a range of 0.8-1.33. This ratio was applied to evaluation of clonal involvement of individual megakaryocytes in the patients with MDS. In two patients with monosomy 7, the majority of the megakaryocytes were monosomic. In the five patients with trisomy 8, all or a majority of the analyzed megakaryocytes were trisomic. These results add direct evidence that in MDS megakaryocytes are involved in the malignant clone. Genes Chromosomes Cancer 25:332-338, 1999.

Favorable outcome after 1-year treatment of childhood T-cell lymphoma/T-cell acute lymphoblastic leukemia.

BACKGROUND: For T-malignancies in children a poor prognosis is reported. In these malignancies a combination of lymphoma and leukemia is commonly seen at presentation and most patients are treated according to protocols for acute lymphoblastic leukemia (ALL). These protocols are often designed for the majority of ALL cases, i.e., progenitor-B-ALL. In pediatric lymphoblastic non-Hodgkin's lymphoma without bone marrow infiltration various protocols have been used. The most frequently reported regimens show variable survival rates between 40 and 75%. PATIENTS AND METHODS: From 1989 we have treated 32 consecutive patients with T-cell malignancies, irrespective of localization, with a protocol consisting of a 4-agent induction treatment followed by high doses of methotrexate, and cytosine-arabinoside and intensified bleomycin, adriamycin, cyclophosphamide, vin cristin, prednisone (BACOP) courses. Treatment duration for each patient was 1 year. Twenty-one of the 32 patients had stage IV disease. Follow-up ranged from 1.6 to 7.6 years (median 4.2 years). RESULTS: Overall event-free survival (EFS) was 72%, while in those with stage IV disease it was 67%. No therapy-related deaths occurred. Neither stage, initial leukocyte value, mediastinal involvement, bone marrow involvement, nor the presence of CD1, CD3, CD4, CD8, or CD10 epitopes was prognostically significant. Evaluation of toxicity revealed a minimal decrease of carbon monoxide diffusion and cardiac shortening fraction. CONCLUSION: A relatively short but intensive chemotherapy can be used in T-cell malignancies. The EFS is satisfying, but larger studies are needed.

A complication of percutaneous tracheostomy whilst using the Combitube for airway control.

We report the occurrence of oesophageal perforation and dilatation during percutaneous tracheostomy. The Combitube was used for airway maintenance during this procedure. This case highlights the limitations of the Combitube when used in this situation.

Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

Prolonged persistence of PCR-detectable minimal residual disease after diagnosis or first relapse predicts poor outcome in childhood B-precursor acute lymphoblastic leukemia.

The follow up of minimal residual disease (MRD) in childhood B-precursor ALL by polymerase chain reaction (PCR) may be of help for further stratification of treatment protocols, to improve outcome. However, the clinical relevance of this approach has yet to be defined. We report the retrospective follow-up of MRD in bone marrow (BM) samples from 50 childhood B-precursor ALL patients by IgH/TCR delta PCR. Twenty-two patients remained in continuous complete remission (median follow-up 61 months), and 28 experienced relapse (median follow-up 75 months). Initial regression of MRD on therapy correlated with outcome. At the end of induction therapy 2/18 (11.1%) patients from the CCR group were PCR positive vs 10/16 (62.5%) from the 'relapse' group (P = 0.005). The presence of PCR detectable MRD predicted event-free survival independent of standard clinical and cytogenetical parameters. Also subsequent to first BM relapse, a correlation between MRD regression and outcome was observed. Six of eight patients who became PCR negative in the time period between relapse and bone marrow transplantation are in CCR, whereas 7/7 patients who remained PCR positive in this time period died (P = 0.006). In approximately 70% of evaluable patients, clinical relapse was preceded by recurrence of detectable MRD at time intervals of 3-18 months earlier and the recurrence of PCR positivity after a period of negativity was always followed by overt relapse. At relapse, the combined use of IgH and TCR delta probes reduced false negativity caused by clonal evolution to approximately 10%. This study shows that the evolution of PCR detectable MRD is an independent predictor of outcome.

Flow cytometric detection of chromosome abnormalities by measuring centromeric index, DNA content, and DNA base composition.

This paper highlights two improvements of the on-line centromeric index (CI) analysis for the detection of chromosome abnormalities. On-line CI versus DNA content analysis of an EBV-transformed cell line, with a deletion (11)(p13p15.1), of a patient with aniridia and Wilms' tumour demonstrates the first improvement of the method of on-line CI analysis for flow karyotyping and sorting; a reciprocal translocation, insertion, or deletion can, when the cell type contains not more than a few of these types of abnormalities, be traced to the p-arm or q-arm of the relevant chromosome. On-line CI analysis was also performed with chromosomes isolated from a transitional cell carcinoma of the bladder. Cytogenetic analysis of this cell line showed numerous chromosomal abnormalities. Chromosomes of this cell line were also karyotyped by bivariate flow cytometry using a different set of parameters: Hoechst 33,258 fluorescence intensity (HOfl) versus chromomycin A3 fluorescence intensity (CAfl). A comparison of these results reveals the second improvement of the CI method for the detection of chromosome abnormalities; bivariate analysis of CI versus propidium fluorescence (PIfl) are complementary to bivariate analysis of HOfl versus CAfl. Chromosomes with distributions that fuse together in the HO/CA flow karyotype may be distinguished as individual peaks on the basis of their CI values.