Immunodiagnosis of childhood all: Problems associated with the use of peripheral blood alone

The immunologic classification of acute lymphoblastic leukemia (ALL) based solely on peripheral blood (PB) cell phenotypes may lead to conflicting results. This was demonstrated by the simultaneous assay of five immunologic markers on PB and bone marrow (BM) cells from 13 children with untreated ALL. We assayed erythrocyte (E) rosettes at 4°C and 37°C, presence of membrane Ig(mIg), and binding of antisera raised against thymus (T), and E^? ALL blasts, respectively. At diagnosis, the PB of these children contained > 90% lymphoid cells with 0–48% E rosettes and 1-84% cells with T antigen(s). Of 7 children with WBC < 10,000/cu mm there were 4 who had 20% or more E rosettes and T-antigen-positive cells. Of 6 children with WBC > 10,000/cu mm there were only 2 who had more than 20% E rosettes and T-antigen-positive cells. Based on examination of PB alone, six children may have been classified as having T-like ALL. However, these results were due to the presence of circulating normal T lymphocytes, and assay of BM cells established that only one of the 13 children had T-like ALL and none had B-cell ALL. Bone marrow blasts from 12 patients did not form rosettes at 37°C, did not have mIg, and did not react with anti-T serum. A high proportion of BM blasts from these 12 patients (39-96%) did react with antiserum against E^? ALL blasts. Of these 12 patients 11 had a higher proportion of E^? ALL antiserum-positive blasts in the BM than PB. Thus, immunologic classification of ALL should be based on the study of BM blasts, or both PB and BM cells.     

The effect of short-course high-dose methylprednisolone on peripheral blood CD34+ progenitor cells of children with acute leukemia during remission induction therapy

This study was undertaken to determine the effect of short-course high-dose methylprednisolone (HDMP) treatment on peripheral blood (PB) CD34+ progenitor cells during remission induction treatment in 11 children with newly diagnosed acute leukemia (7 with ALL, 4 with AML) whose bone marrow (BM) cells expressed fewer than 5% CD34 at the time of diagnosis. All children who had no infection were given HDMP as a single daily oral dose of 30 mg/kg for the first four days of induction therapy. The number of CD34+ progenitor cells were determined by flow cytometry before and after four days of HDMP treatment. While the number of PB blast cells significantly decreased after only a four-day course of HDMP treatment, the number of PB CD34+ progenitor cells increased in all patients. In addition, after four days of HDMP treatment polymorphonuclear leukocytes (PMN) and mononuclear cells (MNC) increased significantly (p < 0.05). We suggest that the potential beneficial effects of HDMP in the induction treatment of acute leukemia may occur partly by the stimulation of PB CD34+ hematopoietic progenitor cells in a short period of time.     

Expression of AC133 vs. CD34 in acute childhood leukemias

AC133, a newly discovered antigen on human progenitor cells, demonstrating 5-transmembranous domains is expressed by 30-60% out of all CD34+ cells. Our aim therefore was to investigate the extent of human stem-/progenitor cells expressing AC133 antigen in umbilical cord blood, peripheral blood without or following an application of granulocyte-colony stimulating factor (rhG-CSF). The main task was the investigation of bone marrow aspirates derived from children suffering from newly diagnosed acute leukemias, as well as from patients with a relapse or during a complete remission. The determination of antigen expression was done by application of flow cytometry (FACScan analysis) and the usage of newly developed monoclonal antibodies (AC133/1 and AC133/2; Miltenyi Biotec GmbH) in combination with monoclonal antibody directed against CD34-antigens (HPCA-2; BD). Our studies till now show average percentages in umbilical cord blood derived from 43 newborns about 0.294 +/- 0.165% AC133+ vs. 0.327 +/- 0.156% CD34+ hematopoietic stem-/progenitor cells (HSPC). In peripheral blood from 11 healthy donors we verified up to 0.15% CD34+ as well as AC133+ HSPC's. The concentration of progenitor cells was found to be obviously higher in peripheral blood from children with various diseases (neuroblastoma, rhabdomyosarcoma, ALL/AML) and undergoing application with rhG-CSF in order to be prepared for PBSC-transplantation. In those cases we found up to 3.51% AC133+ cells as well as slightly higher values (3.94%) for CD34 antigens. Additionally we quantified 128 bone marrow (BM) samples for AC133+ and CD34+ cells. In 10 BM samples, derived from patients without any neoplasia, the CD34+ cells were about 0.03% and 1.49%, whereas AC133 values were up to 0.64%. Bone marrow aspirates from 53 children with acute leukemias at time of diagnosis (ALL: n = 41/AML: n = 12) have been immunophenotyped and leukemic blast cells have been proved for AC133- and CD34 antigen expression. 32/41 (78%) of lymphoblastic leukemic cells showed to be positive for CD34 antigen and 24/41 (58%) demonstrated AC133 antigens. Interestingly there were 2 ALL-patients with pathological blast cells positive for AC133 but lacking of any CD34 antigens. 42% (5/12) of investigated AML patients showed CD34+ phenotype, on the other hand there were only 25% (3/12) with AC133+ phenotype. Similar values were found in relapsed patients (n = 18). In BM samples from patients during complete remission (n = 47) we could detect percentages up to 5.55% for CD34 and up to 1.25% for AC133 positive stem-/progenitor cells. Such quite high data may be explained by occasionally application of rhG-CSF therapy. Our results till now lead to the conclusion, that it seems to be useful, to recruit quantification of CD34+ HPSC by additionally detecting AC133 antigens. This new stem cell marker (AC133) may be of great value in case of autologous peripheral blood stem cell transplantation (PBSCT) because it could be an alternative to the usual CD34+ MACS selection system.     

Monitoring of inosine monophosphate dehydrogenase activity in mononuclear cells of children with acute lymphoblastic leukemia: enzymological and clinical aspects

BACKGROUND: Inosine 5'-monophosphate dehydrogenase (IMPDH; EC1.1.1.205) catalyzes the rate-limiting step in guanine nucleotide biosynthesis, and may play an important role in treatment of patients with antipurines. METHODS: We used an HPLC method to measure the IMPDH activity in peripheral blood and bone marrow mononuclear cells (MNC). IMPDH activities were determined in children who were diagnosed with and treated for acute lymphoblastic leukemia (ALL), and in a group of control children. RESULTS: The median IMPDH activity for control children was 350 pmol/10(6) pMNC/hr (range 97-896; n = 47). No gender or age differences were observed. IMPDH activity at diagnosis of ALL was correlated with the percentage of peripheral blood lymphoblasts (r = 0.474; P < 0.001; n = 71). The median IMPDH activity at diagnosis was 410 pmol/10(6) pMNC/hr (range 40-2009; n = 76), significantly higher than for controls (P = 0.012). IMPDH activity significantly decreased after induction treatment, and during treatment with methotrexate (MTX) infusions (median 174 pmol/10(6) pMNC/hr; range 52-516; n = 21). The activity remained low during maintenance treatment with 6-mercaptopurine (6MP) and MTX, at a significantly lower level than for controls (P < 0.004). One year after cessation of treatment IMPDH activity returned to normal values. CONCLUSION: The decrease of IMPDH activity at remission of ALL seems to be at least partly due to the eradication of lymphoblasts with the type 2 isoform of the enzyme.     

The clinical value of follow-up examinations in childhood T-cell acute lymphoblastic leukemia and T-cell non-Hodgkin's lymphoma

BACKGROUND: The aim of this study was to evaluate the value of follow-up investigations of T-cell acute lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkin's lymphoma (T-NHL), including cerebrospinal fluid (CSF) examination, bone marrow (BM) aspiration, peripheral blood (PB) count, serum lactate dehydrogenase (LDH) and chest X-rays in patients with an initial mediastinal enlargement. PROCEDURE: We reviewed clinical records of all T-ALL patients from 1987 to 2002 and all T-NHL patients from 1977 to 2002, seen at a single institution. RESULTS: Of 48 T-ALL patients, 15 suffered from a relapse, 6 (40%) were asymptomatic at the time of relapse. T-ALL (13/30) with mediastinal enlargement at first diagnosis relapsed versus 2/16 of those without mediastinal enlargement. However, at relapse, only one patient had a mediastinal mass, which in addition was symptomatic. Of 39 T-NHL patients, 6 patients relapsed. Forty percent of relapsed T-ALL and 17% of relapsed T-NHL were asymptomatic. The seven asymptomatic relapses were detected by CSF (n = 4), BM (n = 2) or blood count (n = 1) examinations. All T-ALL and T-NHL patients with a mediastinal relapse were symptomatic. CONCLUSIONS: This study suggests that routine CSF examinations during treatment can detect relapses of T-ALL and T-NHL before onset of symptoms, which might be of clinical value. Relapses are rarely detected by BM or blood examinations and whether this translates in a clinical benefit is unlikely. Routine chest X-rays are not useful.     

Cell-Mediated Cytotoxicity in Children During and after Therapy for Acute Lymphoblastic Leukemia

Cell-mediated cytotoxicity is considered to play a major role in immune defense and in particular in the killing of virus-infected and neoplastic cells. It appears to have some interesting implications when considering the infectious risk of acute lymphoblastic leukemia (ALL) children during immunosuppressive chemotherapy and the role of self-defense against minimal residual disease. We have studied natural killer (NK) activity and lymphokine-activated killer (LAK) activity in children during and after treatment for ALL. We observed that peripheral blood mononuclear cells in 22 children undergoing maintenance chemotherapy displayed significantly depressed NK activity compared with normal controls even when the proportion of NK cells was normal. LAK activity was also considered in 43 ALL children during and after maintenance chemotherapy. We observed that LAK activity was persistently comparable with that of normal controls. It seems definite that NK activity impairment is transient and is completely restored in ALL children a few months after chemotherapy has been successfully completed. The evidence that LAK activity is not impaired in ALL children may have some implications in view of a possible immunomodulatory approach in the presence of refractory disease.     

Hematologic involvement in mitochondrial cytopathies in childhood: a retrospective study of bone marrow smears.

We retrospectively analyzed the bone marrow (BM) smears of 10 children with mitochondrial cytopathies. Light microscopic examination showed large and coalescent cytoplasmic vacuolization of some BM precursors in nine cases, including two children with normal peripheral blood counts and four with sideroblastic anemia. BM ultrastructural study showed abnormal mitochondria in the erythroid lineage in all three children studied. Ultrastructural studies in two cases revealed a population of giant mitochondria with abnormal ultrastructure coexisting with a population of normal mitochondria in proerythroblasts, basophil erythroblasts, and less commonly in more mature erythroblasts. In a third child, mitochondria were normal in size with cristae either absent or exhibiting abnormal longitudinal orientation. Heteroplasmic segregation of mitochondria during cell division could account for the finding of a double population of cells on ultrastructural examination. These features suggest that cytologic and ultrastructural BM examination could be useful for the diagnosis of mitochondrial disorders. That is, when large and coalescent cytoplasmic vacuoles of BM precursor cells are present, the clinician should search for mitochondrial cytopathy in a child with unexplained cytopenia(s).     

Investigation of TT virus in the etiology of pediatric acute lymphoblastic leukemia

Infectious agents have been implicated in the pathogenesis of pediatric acute lymphoblastic leukemia (ALL). A novel human DNA virus, TT virus (TTV), has been identified in children possessing characteristics as an etiologic agent, making the virus a potential candidate. Analysis of specimens from children with ALL was performed to determine if an association exists. Archived specimens (79 peripheral blood mononuclear cells, PBMC/bone marrow cells, BMC, and 125 cerebrospinal fluid, CSF) obtained at diagnosis and during therapy from 28 pediatric patients were tested for TTV. All of the diagnostic BMC/PBMC were negative for TTV, but 7 patients had follow-up specimens that converted to TTV positivity. TTV was not detected in any CSF. The absence of TTV at diagnosis suggests TTV is unlikely to be causally associated with ALL in the cases analyzed. However, the data support TTV transmission through blood products and suggest that the CNS is not a sanctuary site for TTV.     

Bone marrow features in children with HIV infection and peripheral blood cytopenias

HIV infection is associated with numerous abnormalities affecting both the myeloid and lymphoid lineages. We studied the features associated with peripheral cytopenias as the first sign of HIV infection in children. Peripheral blood (PB) counts, PB and bone marrow (BM) lymphocyte subsets, as well as viral load and serum levels of ferritin, vitamin B12, and folic acid were determined. Five children were naive of treatment (Group 1) and three were under HAART (Group 2). In Group 1 all patients had anemia of chronic disease. One had a bone marrow culture positive for Mycobacterium avium intracellulare and pancytopenia. Besides this, neutropenia and thrombocytopenia were seen in one patient each. In Group 2 anemia was found in all, neutropenia in one, and thrombocytopenia in two patients. Peripheral blood cytopenias were due to HAART toxicity in one patient. In the other two they were due to iron or folate deficiency. Bone marrow cytology showed cell abnormalities mainly in granulocytic precursors and megakaryocytes. All except two (taking HAART) patients had a high viral load. There was a straight correlation between viral load in PB and bone marrow. Viral load was correlated with peripheral CD4 but not with CD8 lymphocytes. A decrease in bone marrow B lymphocytes was seen in all patients. The introduction of HAART improved peripheral cytopenias. Bone marrow examination was useful for determining the etiology of the cytopenias and for detection of opportunistic infection. Hemopoietic cell abnormalities were similar to those seen in adults and indicative of HIV infection.     

CXCR4在儿童急性白血病的表达及临床意义

目的探讨儿童急性白血病(AL)骨髓细胞表面CXCR4的表达及其临床意义。方法采用流式细胞术分别检测43例初诊未治急性白血病患儿(实验组)和10例非恶性血液病患儿(对照组)骨髓细胞表面CXCR4的表达情况。结果1.急性白血病患儿骨髓细胞表面CXCR4的相对荧光强度明显高于对照组(P<0.05)。2.ALL组CXCR4的相对荧光强度明显高于AML组(P<0.05)。3.髓外浸润组患儿初诊时CXCR4的相对荧光强度明显高于非髓外浸润组。(P<0.05)。4.实验组患儿CXCR4的相对荧光强度与外周血白细胞计数(WBC)呈正相关关系,相关系数为0.58(P<0.05)。结论1.CXCR4在儿童急性白血病初诊时为高水平表达,提示它可以作为急性白血病的一种检测指标。2.CXCR4的表达在ALL明显高于AML,说明它与急性白血病的类型具有一定相关性。3.急性白血病患儿骨髓细胞表面CXCR4的高表达与髓外浸润密切相关,并且和初诊时高白细胞计数有关。     

Implications of terminal deoxynucleotidyl transferase activity in various forms of childhood leukemia (author's transl)

Terminal deoxynucleotidyl transferase (TdT) was examined in mononuclear peripheral blood cells (pB) and bone marrow (BM) specimens of 63 children with acute leukemia (AL). The enzyme activity in normal specimens (pB, BM) was below 0.2 U/10(8) cells; whereas, 49 of the 52 children with acute lymphoblastic leukemia (ALL) at diagnosis showed an activity in the range 1.2--60 U/10(8) cells. 2 of the remaining 3, devoid of TdT activity, were found to be B-cell leukemia. Patients with acute non-lymphoblastic leukemia (ANLL) were generally TdT-negative. Elevated level of TdT activity was detected in only one of 11 children with ANLL. In one patient with acute leukemia two distinct populations of cells with lymphoblastic (87%) and myeloblastic characteristics were evident. The clinical course and cell marker studies were consistent with the interpretation of a defect at the level of the common stem cell giving rise to a TdT-positive lymphoblastic cell population at diagnosis and, following the initial ALL-therapy (4 weeks), a predominant TdT-negative myeloblastic population. TdT as a marker for the modulation of chemotherapy was examined in the remission phase of the disease. Of the nine patients in the first 3 months of remission, one was found to have elevated level of TdT activity (2.5 U/10(8) cells). These data define the usefulness of TdT in the classification of acute leukemia.     

Daunorubicin-induced cell kill with 1-hour versus 24-hour infusions: a randomized comparison in children with newly diagnosed acute lymphoblastic leukemia

BACKGROUND: Daunorubicin (DNR) is one of the most important drugs in treatment of acute lymphoblastic leukemia (ALL). Prolonged infusions of anthracyclines are less cardiotoxic but it has not been investigated whether the in vivo leukemic cell kill is equivalent to short-term infusions. PROCEDURE: In the cooperative treatment study COALL-92 for childhood ALL 178 patients were randomized to receive in a therapeutic window a single dose of 36 mg/m (2) DNR either as a 1-h (85 patients) or 24-h infusion (93 patients). Daily measurements of white blood cell count (WBC) and peripheral blood smears for seven days could be evaluated centrally in 101 patients (1-h: 43 patients, 24-h: 58 patients). RESULTS: The proportional decline of blasts at day 7 after DNR infusion showed no statistically significant difference between the two treatment arms. At day 3 the median percentage of blasts was less than 10%, at day 7 less than 2% for either the 1-h or 24-h infusion. Twelve patients (1-h: 5 patients, 24-h: 7 patients) had an absolute number of more than 1000 blasts per mul peripheral blood (PB) at day 7 after DNR infusion (DNR poor responders). Kaplan-Meier analysis showed an equal probability of EFS for the short- and long-term infusion group (24-h: 83%+/-5; 1-h: 81+/-6) after a median observation time of 12.3 years. CONCLUSIONS: We conclude that in children with ALL a 24-h infusion of DNR has the same in vivo cytotoxicity for leukemic cells as a 1-h infusion. This offers the possibility to use prolonged infusions with hopefully less cardiotoxicity without loss of efficacy.     

小儿急性淋巴细胞性白血病伴继发骨髓纤维化及疗效观察

对1991年10月至1996年2月收治的74例小儿急性淋巴细胞性白血病（ALL）同时作骨髓（BM）涂片与切片（活检）检查，发现11例伴有继发骨髓纤维化（SMF）。其中5例BM涂片与切片增生度不相符，涂片增生度减低或极度减低，切片示增生活跃至极度活跃（相差2～5个级别）；4例BM涂片未达ALL诊断标准，原淋＋幼淋＜0．3（0．08～0．25），切片见大量幼稚细胞浸润；6例外周血白细胞＜3×109/L（1．3～2．79×109/L）。4例早期用VLP方案为主治疗，持续完全缓解时间为16～30个月，疗效良好。2例早期用VCP方案治疗，一直未能完全缓解。BM切片示脂肪组织增多（70％和75％），疗效差。结果表明：BM切片与涂片结合对小二ALL伴SMF的诊断有重要价值；小儿ALL伴SMF的发生率可能不低；其治疗的成功与否与化疗早期的选择关系密切。     

影响儿童急性淋巴细胞白血病预后的多因素分析

目的探索影响儿童急性淋巴细胞白血病(ALL)长期生存的预后因素。方法采用回顾性队列研究,对1998年1月1日至2003年7月1日我院小儿血液科就诊,治疗时间大于6个月,年龄小于15周岁且能够按时随访的ALL初诊患儿进行生存分析。结果76例患儿总诱导缓解率为94.7%,中位生存时间86个月,5年无事件生存率(5-EFS)为(70±1.23)%。合并数据COX回归多因素生存分析显示:初诊时外周血白细胞数50×109/L,血红蛋白含量(60 g/L,肝肋下≥5 cm,泼尼松诱导不敏感,诱导35天骨髓原始、幼稚淋巴细胞55%,治疗不依从,治疗过程中复发,均显著增加ALL儿童生存风险。结论初诊时外周血白细胞数和血红蛋白水平,肝脏肋下大小,35天骨髓原始、幼稚淋巴细胞比例,泼尼松诱导试验,治疗过程中复发,治疗依从性是影响ALL患儿长期生存的独立预后因素,提高治疗的依从性和个体化治疗对提高ALL儿童生存率有重要作用。     

儿童急性淋巴细胞白血病T细胞受体重排删除环与严重感染相关性研究

目的：定量检测急性淋巴细胞白血病（急淋）患儿不同阶段外周血中信号连接/结合T细胞受体重排删除环（sjTRECs）水平的变化,以评价其作为预测化疗后严重感染指标的可行性。方法：共收集初发急淋患儿（初发组,n=30）、早期强化化疗结束后白细胞恢复且未感染患儿（化疗组,n=36）、化疗后严重感染患儿（感染组,n=30）及正常同龄儿童（正常组,n=50）外周血标本共146份,通过FQ PCR法检测其sjTRECs水平,并进行组间比较。结果：正常组平均sjTRECs为（394±270）copies/103 MNC,高于其他3组（P<0.05）;化疗组sjTRECs水平低于初发组 ［(96±78) copies/103 MNC vs (210±219）copies/103 MNC,P<0.05］;感染组sjTRECs水平最低,仅为（48±40） copies/103 MNC。结论：监测急淋患儿化疗后sjTRECs水平变化可能有助于早期预测机体严重感染的发生。     

Evaluation of Early Marrow Response in Childhood Aneuploid Acute Lymphoblastic Leukemia: Flow Cytometric DNA Analysis versus Standard Morphology

Despite improved survival rates for childhood acute lymphoblastic leukemia (ALL), the relapse rate remains at 20–30%. Early peripheral blood and bone marrow (BM) responses have been associated with more favorable outcomes; all current childrens cancer group (CCG) protocols for ALL require BM evaluation at days 7 and 14 with subsequent therapy based on the results. Morphologic interpretation of aspirate smears during induction chemotherapy is challenging, as the samples are often hypocellular, excessively friable, and cytologically altered by drugs. We have shown discordancy of day 7 and 14 BM lymphoblast counts using morphologic and flow cytometric immunophenotypic analyses (FC). The aim of our study was to determine the utility, reliability, and cost effectiveness of lymphoblast enumeration using DNA content analysis by flow cytometry (DNA-FC) and to further demonstrate the subjectivity of morphologic review. All new cases of ALL had DNA-FC and FC analyses. The percentage of lymphoblasts as determined by both methods was compared for 82 aneuploid cases. Three pathologists independently reviewed aspirate smears from 39 bone marrow samples of aneuploid ALL that were obtained during early induction. These results were compared among themselves and with the results obtained by DNA-FC. We found excellent correlation between the percentage of lymphoblasts as determined by DNA-FC and FC (R ² = 0.97) over a range of 0 to 99%. Pathologic review agreed with the DNA-FC, on average, 68%. The sensitivity, specificity, and positive and negative predictive values of morphologic review, averaged 53, 84, 78, and 63%, respectively, when using DNA-FC as the gold standard. All three pathologists achieved agreement of lymphoblast percentage by morphology in 72%. In our laboratory, the use of DNA-FC equates to one-sixth the time and one-half the price of FC per exam. We have shown a strong correlation between blast counts determined by DNA-FC and FC. DNA-FC is an objective, economical, and reliable method to assess early response in induction marrows from aneuploid ALL where morphology is often uninterpretable. This test is highly reproducible and available at most pediatric institutions. Prospective studies need to be employed to evaluate the effect of more definitive methods (DNA-FC and FC) of assessing the early response in bone marrows on prognosis.