Importance of FISH combined with Morphology,Immunophenotype and Cytogenetic Analysis of Childhood/ Adult Acute Lymphoblastic Leukemia in Omani Patients

Genetic changes associated with acute lymphoblastic leukemia (ALL) provide very important diagnostic andprognostic information with a direct impact on patient management. Detection of chromosome abnormalities byconventional cytogenetics combined with fluorescence in situ hybridization (FISH) play a very significant rolein assessing risk stratification. Identification of specific chromosome abnormalities has led to the recognition ofgenetic subgroups based on reciprocal translocations, deletions and modal number in B or T-cell ALL. In the lasttwelve years 102 newly diagnosed childhood/adult ALL bone marrow samples were analysed for chromosomalabnormalities with conventional G-banding, and FISH (selected cases) using specific probes in our hospital.G-banded karyotype analysis found clonal numerical and/or structural chromosomal aberrations in 74.2% ofcases. Patients with pseudodiploidy represented the most frequent group (38.7%) followed by high hyperdiploidygroup (12.9%), low hyperdiploidy group (9.7%), hypodiploidy (<46) group (9.7%) and high hypertriploidygroup (3.2%). The highest observed numerical chromosomal alteration was high hyperdiploidy (12.9%) withabnormal karyotypes while abnormal 12p (7.5%) was the highest observed structural abnormality followed byt(12;21)(p13.3;q22) resulting in ETV6/RUNX1 fusion (5.4%) and t(9;22)(q34.1;q11.2) resulting in BCR/ABL1fusion (4.3%). Interestingly, we identified 16 cases with rare and complex structural aberrations. Application ofthe FISH technique produced major improvements in the sensitivity and accuracy of cytogenetic analysis withALL patients. In conclusion it confirmed heterogeneity of ALL by identifying various recurrent chromosomalaberrations along with non-specific rearrangements and their association with specific immunophenotypes. Thisstudy pool is representative of paediatric/adult ALL patients in Oman.     

Molecular and Cellular Correlates of Methotrexate Response in Childhood Acute Lymphoblastic Leukemia

The improved outlook for children diagnosed today with acute lymphoblastic leukemia (ALL) over that 40 years ago is remarkable. With modem therapies and supportive care, complete remissions are achieved in up to 95% of patients and long-term disease-free survival rates approach 80%. Methotrexate is a key component in ALL consolidation and maintenance therapies and is administered intrathecally in the prophylaxis and treatment of central nervous system leukemia. Recent reports have significantly extended the results of preclinical studies of methotrexate response and resistance to patients with ALL. The application of new and sensitive molecular biology techniques makes it possible to study specific chromosomal and genetic alterations[t(12;21), hyperdiploidy, deletions or methylation of p15^INK4B and p16^INK4A] which potentially contribute to methotrexate response and resistance in childhood ALL. Studies of the relationships between genetic alterations and ALL progression, methotrexate pharmacology, and long term event-free-survivals may lead to the better identification of subgroups of patients who exhibit unique levels of sensitivity or resistance to chemotherapy including methotrexate. Further, by characterizing the roles of translocation-generated fusion genes (TEL-AML1) and tumor suppressor genes (p15^INK4B and p16^INK4A) in treatment response, it may be possible to identify new and selective targets and/or treatment strategies for both children and adults with ALL who are refractory to current therapies.     

Acute Myeloid Leukemia with Translocation (8;21). Cytomorphology, Dysplasia and Prognostic Factors in 41 Cases

The translocation t(8;21) is one of the most common structural aberrations in acute myeloid leukemia (AML). Excellent response rates and a better relapse-free survival have been described. We analyzed specific morphologic and cytochemical features including dysplasia and other prognostic factors in 41 patients with AML and t(8;21) who underwent aggressive chemotherapy in two national cooperative group studies. Five patients were classified as AML M1 and 36 as AML M2 according to the FAB criteria. Auer rods were detected in 28 patients (68%), however in only 16 patients were they “thin and elongated” as has been described as typical for t(8;21). The presence or absence of Auer rods did not appear to be associated with disease-free survival in this sample. Dysgranulopoiesis was detected in 31/41 patients (90%); five of these patients additionally had dyserythropoiesis (12%). In six cases (15%), dysmegakaryopoiesis was seen in combination with dysgranulopoiesis. Only one patient had trilineage dysplasia. Dysplastic features had no influence on prognosis. Additional cytogenetic abnormalities were detected in 24/41 patients. Twelve male (48%) and four female (25%) had a loss of a sex chromosome. This was correlated with a better disease-free survival (p = 0.039). The complete remission rate (CR) to chemotherapy was 90%. The early death rate was 10%. Disease-free survival of the complete responders was 60% at two years with no relapses observed in ten patients with 2-6 years of follow up. This favorable disease-free survival was observed with a variety of post-induction regimens and t(8;21) had been detected as an independent factor for good prognosis. The need for very intensive therapy, such as bone marrow transplantation, is unanswered at this time.     

Methotrexate Pharmacology and Resistance in Childhood Acute Lymphoblastic Leukemia

Impressive gains have been made in the therapy of childhood acute lymphoblastic leukemia (ALL) in recent years such that remissions today are commonly achieved in up to 95% of patients and long term disease-free survival rates approach 70%. Methotrexate is a key component in ALL consolidation and maintenance therapies and is administered intrathecally in the prophylaxis and treatment of central nervous system leukemia. Critical determinants of methotrexate sensitivity and resistance (di-hydrofolate reductase levels, methotrexate membrane transport, methotrexate polyglutamylation) previously described in cultured cells have recently been identified in lymphoblasts from children with ALL. Heterogenous expressions of increased dihydrofolate reductase or impaired methotrexate transport can be detected in both diagnostic and relapsed ALL specimens by flow cytometry with fluorescent methotrexate analogues. Lymphoblasts from children with ALL synthesize long chain polyglutamates and correlations have been established between the accumulation of methotrexate polyglutamates in ALL blasts and characteristic patient prognostic features. Variations in methotrexate polyglutamate accumulation may reflect changes in polyglutamate synthetic or degradative enzymes, or may be secondary to changes in methotrexate influx or dihydrofolate reductase levels. Other critical elements in treatment response to methotrexate include the dose and route of methotrexate administration, its catabolism to 7-hydroxymethotrexate, and the rate of methotrexate plasma clearance. A unique relationship exists between chromosome 21 and ALL leukemogenesis, and response to treatment including methotrexate. A better understanding of the molecular bases of methotrexate response and the development of methotrexate resistance in childhood ALL should facilitate further improvements in the effectiveness of methotrexate-based chemotherapy for this disease.     

DNA Image Analysis in Childhood Acute Lymphoblastic Leukemia

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid-8 hyperdiploid and 1 hypodiploid.

The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.     

Analysis of Natural Killer Cell Defects in Childhood Acute Lymphoblastic Leukemia

The natural killer (NK) activity of circulating lymphocytesof 38 children with acute lymphoblastic leukemia (ALL) in completeremission was studied by ³¹Cr-release cytotoxicity assay. NK.activity of the non-T-cell fraction (NK_non-T) and of the T-cellfraction (NK_T) was also measured. In addition, the number ofcirculating NK cells was determined by an immunofluorescencetechnique using monoclonal anti-Leu-7 (HNK-1) antibody. TotalNK activity of the ALL patients in complete remission was impairedduring maintenance therapy (P<0.001) and after cessationof therapy (P<0.05). NK activity of NK_non-T and NK_T cellswas equally impaired (P<0.05). The numbers of Leu-7⁺ cellsand NK activity units were decreased during maintenance therapy(P<0.05), but approached normal after cessation of therapy.These data indicate that decreased NK activity of childhoodALL in complete remission during maintenance therapy is dueto a decrease in total NK cells and impaired function of bothNK_non-T and NKT cells.     

Hypercalcemia in a Case of Childhood Acute Lymphoblastic Leukemia

Severe hypercalcemia (serum calcium, 4.25–5.25 mmol/l),in association with osteolytic bone lesions, was found in agirl aged 2 yr 7 mo with common acute lymphoblastic leukemia(ALL). Hormonal studies excluded the possibility of the hypercalcemiabeing caused by primary hyperparathyroidism or ectopic parathyroidhormone secretion. Increased plasma prostaglandinE₂ (PGE₂).Jlevels (130 ng/l), probably produced by leukemic cells, wereconsidered to be one of the pathogenic mechanisms responsiblefor the occurrence of hypercalcemia in this patient. Both thehypercalcemia and the abnormal plasma PGE₂ level returned tonormal after chemotherapy.     

Diagnostic and Prognostic Utility of Fluorescence In situ Hybridization (FISH) Analysis in Acute Myeloid Leukemia

Purpose of Review

Acute myeloid leukemia (AML) is a hematologic neoplasia consisting of incompletely differentiated hematopoietic cells of the myeloid lineage that proliferate in the bone marrow, blood, and/or other tissues. Clinical implementation of fluorescence in situ hybridization (FISH) in cytogenetic laboratories allows for high-resolution analysis of recurrent structural chromosomal rearrangements specific to AML, especially in AML with normal karyotypes, which comprises approximately 33–50% of AML-positive specimens. Here, we review the use of several FISH probe strategies in the diagnosis of AML. We also review the standards and guidelines currently in place for use by clinical cytogenetic laboratories in the evaluation of AML.

Recent Findings

Updated standards and guidelines from the WHO, ACMG, and NCCN have further defined clinically significant, recurring cytogenetic anomalies in AML that are detectable by FISH.

Summary

FISH continues to be a powerful technique in the diagnosis of AML, with higher resolution than conventional cytogenetic analysis, rapid turnaround time, and a considerable diagnostic and prognostic utility.

     

Bone Mineral Density in Survivors of Childhood Acute Lymphoblastic Leukemia

Background: The objective of this study was to evaluate bone mineral density (BMD) after completion of treatment for childhood acute lymphoblastic leukemia (ALL). Methods: In this cross-sectional study, 103 survivors of ALL aged 13.5 ± 0.45 who completed their treatment at least one year earlier were enrolled. Among these, 49.5% and 51.5% received chemotherapy alone and chemotherapy plus cranial radiotherapy, respectively. Bone mineral content, BMD, and bone mineral apparent density in the lumbar spine (LS), femoral neck (FN) and forearm were assessed using dual-energy X-ray absorptiometry (DEXA). BMD Z-scores were classified according to International Society for Clinical Densitometry (ISCD) criteria. Results: The mean BMD Z-scores ± SD forLS, FN and forearm were -1.60 ± 0.12, -1.21 ± 0.9 and -2.43 ± 0.14 respectively with significant differences (P<0.001). Considering the lowest BMD Z-score in LS and FN areas (at any site) and according to the ISCD classification, 62.1%, 33% and 4.9% of the patients had normal BMD, low BMD and osteoporosis, respectively. Also, 8.7% of patients had developed fractures after completion of the treatment period, 4.9% having BMD Z-Scores Conclusions: ALL patients are at risk for low BMD and fracture. Therefore, applying DEXA scanning is recommended after completion of therapy for prevention of BMD reduction and osteoporosis.     

Skewed Rearrangement of the VH4-21 Gene During Pre-B Acute Lymphoblastic Leukemia

Thirty-six pre-B acute lymphoblastic leukemias (ALL) were studied for VH family expression. Among the 35 detected rearrangements, VH1 family genes were expressed in 7, VH2 in 1, VH3 in 18, VH4 in 6 and VH6 in 3. This expression is close to that expected according to the complexity of the system. The complete sequence of the 6 VH4 genes was examined in order to determine whether there is a skewed rearrangement of individual genes in this family. Our results indicate rearrangement of VH4-21 in 3 cases, 71 -4 in one, 58P2 in one case and probably of a new germinal VH4 gene for the sixth case. All the genes were displaying an almost complete homology with their germinal VH counterparts. The 6 sequenced genes associated with 6 different D gene segments displaying a close homology with their germinal counterpart. JH4 segment was expressed in 3 cases and JH6 in the remaining 3. These results associated with previous results obtained by others indicate that there is skewed rearrangement of the VH4-21 gene in pre-B ALL. It is presently unknown whether this phenomenon is the consequence of a selective process or whether it reflects what normally occurs in the normal human functional repertoire, which could be more limited than the germline repertoire.     

Combined Study of Cytogenetics and Fluorescence in Situ Hybridization (FISH) Analysis in Childhood AcuteLymphoblastic Leukemia (ALL) in a Tertiary Cancer Centre in South India

FISH is one of the most sensitive molecular methods to detect genetic abnormalities with DNA probes.When cytogenetic studies are normal or insufficient, FISH may detect cryptic rearrangements, rare or slowlyproliferative abnormal populations in non-mitotic cells. We cytogenetically evaluated 70 childhood ALL - 67.1%were found to have an abnormal karyotype. The 23 patients (32.9%) with a normal karyotype were analyzedby FISH applying two probes; TEL/AML1 and MYB which detect cryptic rearrangements of t(12;21)(p13;q22)and deletion of (6q) respectively, associated with a good prognosis. Out of 23 patients, one was positive fort(12;21)(p13;q22) (4.3%). None of our patients were positive for MYB del(6q). Two patients showed an extrasignal for MYB on chromosomes other than 6 (8.6 %) indicating amplification or duplication. Findings werecompared with the available literature. Our study clearly indicated the integrated FISH screening method toincrease the abnormality detection rate in a narrow range. FISH is less useful for diagnostic study of patientswith suspected del(6q) but it helps in detecting known cryptic rearrangements as well as identification of newabnormalities(translocation , duplication and amplification) at the gene level.     

Telomere Reduction of Specific Chromosome Translocation in Acute Myelocytic Leukemia

The length of telomere restriction fragments (TRF) was studied by Southern blotting in 42 patients with acute myelocytic leukemia (AML) including 15 patients with 8;21 translocation, 8 with 15;17 translocation and 19 with a normal karyotype. The TRF length of leukemic cells with a normal karyotype and with 8;21 or 15;17 translocation showed significant reduction compared to that of lymphocytes from normal individuals (P<0.05). In addition, there was a statistically significant difference in TRF length between leukemic cells with a normal karyotype and 8;21 translocation (P<0.05). Therefore, the significant difference of telomere reduction in 8;21 translocation may be an important factor in the leukemogenic process.     

T-Cell Acute Lymphoblastic Leukemia with t(11;14) in Children

We review and update our examination of the clinical and biologic findings in 19 cases of acute lymphoblastic leukemia (ALL) with the t(11;14) and discuss the literature relevant to the clinical, biologic, and molecular aspects of these translocations. In nine consecutively diagnosed cases at St. Jude Children's Research Hospital and 10 cases reported by other institutions, clinical features did not differ among T-cell ALL patients with and without the t(11;14), although leukemic cells with this translocation were more likely to coexpress CD4 and CD8 antigens. The t(11;14)(p13;q11) appears to occur exclusively in T-cell malignancies of intermediate- or late-stage thymocyte differentiation; further studies will be needed to determine whether it has prognostic significance.     

Acute Complications and Survival Analysis of Childhood Acute Lymphoblastic Leukemia: A 15-year Experience

BackgroundWe evaluated the acute complications that occurred during the treatment of childhood acute lymphoblastic leukemia (ALL) and documented the survival rates of children with ALL.Materials and MethodsWe retrospectively evaluated 110 children with a diagnosis of ALL treated with the Children’s Oncology Group protocol from 1999 to 2014. The demographic, clinical, and laboratory data of 110 patients and acute complications of eligible and evaluable 105 patients were recorded.ResultsOf the 110 patients, 65 were male and 45 were female. The mean age at admission was 8.3 ± 5.2 years. Ninety-seven patients (88.2%) had been diagnosed with pre–B-cell ALL, 11 (10%) with T-cell ALL, 1 (0.9%) with mixed phenotype acute leukemia, and 1 (0.9%) with mature B-cell acute leukemia. Of the 110 patients, 40 (36.3%) were in the standard-risk group and 70 (63.7%) were in high-risk group. Of the 110 patients, 105 had been followed up regularly and evaluated for acute complications. Infection was the most common complication (n = 93; 88.5%), followed by gastrointestinal (n = 29; 27.6%), neurologic (n = 28; 26.6%), metabolic/endocrine (n = 16; 15.2%), drug-related hypersensitivity (n = 16; 15.2%), avascular necrosis (n = 13; 12.3%), thrombotic (n = 11; 10.4%), severe psychiatric (n = 2; 1.9%), and various other (n = 12; 11.4%) complications. Of the 110 patients, 98 were assessed in terms of survival analysis. The 5- and 10-year overall survival rates were both 85.9% (standard error [SE], 3.6%). The relapse-free survival rates at 1, 3, and 5 years were 97.9% (SE, 1.5%), 91.3% (SE, 3%), and 86.3% (SE, 3.7%), respectively.ConclusionChildhood ALL, although categorized as curable malignancy owing to the improvements in treatment strategies in recent years, can cause acute complications affecting various systems. Thus, patients should be treated and followed up by multidisciplinary medical teams with high expertise.     

Implementation of Medication Safety Practice in Childhood Acute Lymphoblastic Leukemia Treatment

Objective: Medical Safety Practice (MSP) is a safe procedure in medication process. It is important to investigatethe use of MSP among childhood cancer patients because pediatric oncology is a high-risk area for potentially harmfuladverse events. The purpose of this study is to determine the effects of the implementation of MSP in chemotherapyon the incidence of medication errors in childhood ALL patient at Dr. Sardjito Hospital, including in 1) transcribing,2) administering, 3) monitoring, 4) the incidence of adverse drugs events. (ADEs). Methods: The study design is aquasi-experimental study with pre- and post-intervention without control. The sample consists of ALL patients whoare taken care of at an academic hospital in Indonesia from 2012 to 2013. The sample was consecutively collectedduring the period of study. The data were collected through medical records, research form, observation, and discussionwith the nurse. The intervention given is training and implementation of medical safety practice in chemotherapy.Result: Based on the analysis of the effect of the implementation of MSP (75 and 106 medical records of pre- andpost-intervention), it is obtained: 1) the adherence of chemotherapy transcribing post-intervention increases significantlycompared to pre-intervention (p<0.05), 2) the adherence of chemotherapy administering increases significantly inalmost every aspect (p<0.05), except in preparing drugs by two different health worker, patient’s confirmation ofADEs management, and verification of drug’s expired date, 3) The adherence of chemotherapy monitoring improvedsignificantly post-intervention (p<0.05), 4) Adverse Drug Events (ADE) decreased significantly post-intervention(p<0.05), from 52.1% to 30.5%. Conclusion: The implementation of MSP decreased the incidence of medicationerrors in ALL patients at Dr. Sardjito Hospital in ordering, dispensing, transcribing, administering, and monitoringchemotherapy. It also reduced the incidence of ADEs related to chemotherapy. Specific training for nurses are neededin order to improve the knowledge and skills, especially for medication error and skill in patients’ care.     

p73基因失活在儿童急性淋巴细胞白血病发病和预后中的意义

目的 探讨p73基因mRNA失表达与儿童急性淋巴细胞白血病(ALL)的发病和预后的关系。方法 采用反转录聚合酶链反应(RT-PCR)方法检测了34例ALL患儿骨髓单个核细胞内p73 mRNA的表达情况，并分析了p73 mRNA失表达与化疗的反应及复发的关系。结果 儿童ALL患者中p73 mRNA失表达率达26．5％(9/34)，且与诱导缓解治疗的反应和复发显著相关(P＜0．05)。结论 p73m RNA失表达在小儿ALL的发病过程中起重要作用，p73 mRNA检测对评估儿童ALL预后，指导临床治疗有一定临床意义。     

Bioavailability and Pharmacokinetic Features of Etoposide in Childhood Acute Lymphoblastic Leukemia Patients

The bioavailability and pharmacokinetic characteristics of etoposide were studied in 12 relapsed B-lineage acute lymphoblastic leukemia (ALL) patients after both intravenous (i.v.) infusion and oral administration. Following a 1 hour i.v. infusion of SO mg/m² etoposide, the elimination half-life ranged from 49.8 min to 509.4 min (mean ± SD = 218.6 ± 134.7 min), the MRT ranged from 71.8 to 734.9 min (mean ± SD = 315.4 ± 194.3 min) and the systemic clearance of etoposide ranged from 15.7 to 38.0 ml/min/m² (mean ± SD = 24.1 ± 7.0 ml/min/m²). The AUC ranged from 2234.9 to 5427.0 μM±min) (mean ± SD = 3827.8 ± 1069.5 μM±min) and V_c ranged from 2026.9 to 13505.2 rnl/m² (mean ± SD = 6825.4 ± 3278.5 ml/m²). The maximum plasma etoposide levels ranged from 6.0 to 28.4 μM (mean ± SD = 13.6 ± 6.3 μM). The bioavailability of oral etoposide was determined by comparing the AUC following i.v. infusion to the AUC following oral administration in the same patient. The overall bioavailability (mean ± SD) was 60.6 ± 22.4% (ranged from 17.6% to 91.2%). The elimination half-life following oral administration (mean ± SD) was 209.8 ± 196.3 min (ranged from 51.0 to 794.2 min). The time required to reach the maximum plasma etoposide concentration was 145.4 ± 118.7 min (ranged from 23.7 to 396.9 min). To our knowledge, this is the first report concerning the bioavailability of etoposide in pediatric leukemia patients. All of the other pharmacokinetic properties of etoposide in pediatric B-lineage ALL leukemia patients reported here were similar to those described previously.     

Fluorescence in Situ Hybridization (FISH) Is a Reliable Diagnostic Tool for Detection of the 9; 22 Translocation

The fluorescence in situ hybridization (FISH) technique for detection of the 9;22 translocation was compared with the “gold standard” of conventional cytogenetics. For this purpose, both methods were applied to 81 bone marrow aspirates and/or peripheral blood specimens comprising 50 CML cases and controls from 31 patients without CML. Independently, core biopsies of these 81 patients were investigated by three histopathologists. Conventional karyotype analysis from unstimulated bone marrow cells was successful in 71/81 cases and demonstrated the Ph-chromosome in 42/46 CML patients. With FISH, results were obtained in all 81 cases investigated, confirming fusion of the bcr and abl genes in all cytogenetically Ph-positive patients. Among the five Ph-chromosome-negative specimens bcr/abl fusions were detected in only one patient. The percentage of cells found to be Ph-positive by both methods was correlated, but in individual cases considerable differences in the numbers of Ph-positive cells were observed. Different results may be due to selection of cells after in vitro cultivation predominantly.

FISH proved to be a very reliable technique for specimens that do not contain dividing cells. With FISH, large numbers of cells can easily be scored which is an advantage compared to conventional cytogenetics. Therefore, this method is particularly suitable for those whose therapy is being monitored or a relapse is suspected. However, the FISH results should be evaluated critically with respect to the practical limit of sensitivity since non-specific fusion signals can also be observed in a small percentage of cells in non-CML cases. It is suggested that each laboratory define its own threshold of bcr/abl fusion signals for diagnosing Ph-positive CML by FISH.     

左旋门冬酰胺酶对儿童急性淋巴细胞白血病和非霍杰金淋巴瘤胰腺功能的影响

 目的 观察左旋门冬酰胺酶（L-ASP）对儿童急性淋巴细胞白血病和非霍杰金淋巴瘤胰腺功能的影响。方法 VDLP方案治疗136例患儿,观察L-ASP使用前后血、尿淀粉酶,血脂肪酶,血、尿糖,尿酮体变化情况。结果 发生不良反应12例,其中急性胰腺炎（AP）5例,高血糖、糖尿病5例,急性胰腺炎＋高血糖2例。结论 急性胰腺炎、高血糖为L-ASP使用时胰腺副作用的主要表现形式,与患儿年龄、性别相关,严重时可危及生命,须引起高度重视。