Vacuolated neuroblastoma cells mimicking FAB L(3) lymphoblasts in bone marrow aspirates

Abundant cytoplasmic vacuolation of neuroblasts has been noted on bone marrow aspirate (BMA) smears of two patients with metastatic neuroblastoma. Occasional tumor cells were dispersed as individual cells as well as in clumps. These cells had basophilic cytoplasm and several nucleoli, reminiscent of L(3) lymphoblast morphology. Flow cytometric analysis of the bone marrow mononuclear cells and neuron-specific enolase staining of the bone marrow biopsy samples further distinguished the cells as neuroblasts. Cytoplasmic vacuolations of neuroblasts may be a feature of metastatic neuroblastoma cells in BMA smears.     

Diagnosis of neuroblastoma metastasis in bone marrow with a panel of monoclonal antibodies

Neuroblastoma, along with rhabdomyosarcoma, Ewing's sarcoma, and acute lymphoblastic leukemia/lymphoma, is one of the small, round-cell tumors of childhood. All of these malignancies show a propensity to metastasize to bone marrow. Occasionally when the clinical picture is unclear and the tumor is particularly anaplastic, it can be difficult to arrive at a diagnosis by conventional histological and biochemical procedures. In the present study, a panel of nine monoclonal antibodies was used to undertake a detailed analysis of seven bone marrows contaminated with tumor cells: six cases of stage IV neuroblastoma, and one case of stage IV-S neuroblastoma. The antibody profiles obtained were compared with those deduced from the studies of over 20 marrows from patients with acute lymphoblastic leukemia. A comparison of these data with those obtained from the studies of rhabdomyosarcoma and Ewing's sarcoma cell lines and tissues suggests that when high levels of tumor cells are present in the marrow, it is possible to obtain a confident diagnosis of either neuroblastoma or acute lymphoblastic leukemia. In addition, the immunocytological identification of neuroblasts in bone marrow enables accurate staging without histological examination.     

Detection of residual cells contaminating the bone marrow in neuroblastoma. Apropos of 80 bone marrow evaluations

Eighty bone marrow studies (each including 4 aspirates and 4 trephine biopsies) were performed in 37 children with stage IV neuroblastoma to assess the most accurate means for detection of invasion by neuroblastoma cells. Among 38 abnormal results, only trephine biopsy(ies) were found positive in 24 cases (63%), only aspirate(s) in 5 cases (13%), and both in 9 cases (24%). In 37% of abnormal results, only 1 of the 8 tests performed was found positive. No benefit was obtained from either associated touch imprints of iliac biopsies (67 investigations), or exploration of extra-iliac sites (66 sternums, 53 tibias). Two monoclonal antibodies, claimed to be specific for detection of neuroblastoma cells in bone marrow, were used in 56 investigations; they could detect minimal residual disease in some cases, but they were unreliable when no staining was obtained if initial phenotype of neuroblastoma had not been assessed, or when few isolated cells were observed. Prospective studies using immunocytology and immunohistology are thus warranted.     

神经母细胞瘤骨及骨髓转移模型的建立

目的建立一种裸鼠神经母细胞瘤骨及骨髓转移模型,探讨其在靶向给药系统中的应用价值。方法 4～6周龄雌性BALB/c裸鼠,从股骨远端向骨髓腔注人神经母细胞瘤LA-N-5细胞悬液5μl(约1×105个细胞)制备神经母细胞瘤骨、骨髓转移模型,观察小鼠生存率、肿瘤局部生长及远处转移情况。结果模型成瘤率100%,LA-N-5细胞进入骨髓腔后抑制正常骨髓细胞生长,向骨皮质侵袭性浸润,形成骨溶解灶,并向淋巴结、肝、肾转移。结论股骨内注射人神经母细胞瘤LA-N-5细胞悬液可以成功制作裸鼠神经母细胞瘤骨、骨髓转移模型,后者能较好模拟人神经母细胞瘤在骨骼微环境中的生长及转归情况,是研究神经母细胞瘤骨髓、骨转移及评价临床前靶向给药的适宜模型。     

N-myc status of neuroblastoma from bone marrow cells

The analysis of the N-myc gene in bone marrow specimens at the time of initial diagnosis and as well at the time of relapse from patients with Neuroblastoma stage IV and bone marrow infiltration could give some informations about the N-myc status of these patients. In stage IV neuroblastoma patients with bone marrow infiltration an estimation of the N-myc gene amplification should be attempted, if otherwise no information about the tumor content of the N-myc gene could be gathered. In our investigation we could demonstrate a Southern-blot-analysis of 27 bone marrow specimens with respect to the N-myc gene status which correlated qualitatively well to the N-myc amplification detected later on in the corresponding tumor tissue. In six cases the tumor infiltrated bone marrow showed a clear amplification of the N-myc gene. Because of the contamination by non malignant cells in bone marrow there was a quantitative difference in the calculated N-myc gene copies between the examined bone marrow specimens and corresponding tumor tissue.     

神经母细胞瘤骨髓转移细胞体外原代培养技术的实验研究

目的：根据神经母细胞瘤(neuroblastoma，NB)易发生早期骨髓转移的特点，建立NB骨髓转移细胞体外原代培养的实验技术。方法：对27例伴骨髓转移的NB患者进行NB骨髓转移细胞体外原代培养，并与已建立的SMS-KCNR和SH-SY5Y细胞系进行比较，在倒置相差显微镜下观察细胞生长情况及形态学特点，同时应用单克隆抗体F3系列检测原代培养的NB骨髓转移细胞。结果：在27例NB骨髓转移细胞体外原代培养中，培养成功9例，其细胞生长情况及形态学特点与SMS-KCNR和SH-SY5Y细胞系基本一致，单克隆抗体F3系列检测均阳性。结论：NB骨髓转移细胞体外原代培养技术的成功建立为NB体外诱导逆转及多药耐药等复杂机制研究提供更接近体内生物学性状的细胞来源。     

Autologous bone marrow transplantation. A maximal therapy design for disseminated neuroblastoma

Patients with disseminated neuroblastoma are considered a poor-risk category, hence, our approach towards their treatment should be reconsidered in terms of the unique clinical and biological characteristics of neuroblastoma tumor growth. To this end, we have devised a treatment program consisting of surgery, and a schedule of sequentially escalating doses of cyclophosphamide combined with other drugs until a minimal disease status is obtained. When this is achieved, the patient is treated with maximal therapy, i.e., total body irradiation, high-dose L-phenylalanine mustard and dianhydrogalactitol followed by reconstitution with an autologous bone marrow graft. Details of this program include problems associated with evaluation of response, i.e., evaluation of risk, determination of minimal tumor burden, avoidance of toxicity, and compensation for supportive measures during maximal therapy. Additional problems of purging bone marrow of tumor cells are considered.     

Evaluation of bone marrow metastasis of neuroblastoma and changes after chemotherapy by MRI

To evaluate the usefulness of MRI for diagnosing bone marrow metastasis of neuroblastoma, we compared MRI findings with histological findings. MRI was performed 26 times in 20 patients with neuroblastoma to detect metastasis to the bone marrow of the femur and tibia. Abnormal areas observed by MRI were histologically examined. The lesion visualized by MRI as a low-intensity area on T1-weighted images and as a high-intensity area on T2-weighted images was histologically confirmed to be neuroblastoma in 81% (17/21). The percentage varied according to the treatment state: 89% (8/9) by MRI imaging performed before the initiation of chemotherapy, 67% (6/9) within 3 weeks after cessation of chemotherapy (during chemotherapy), and 100% (3/3) in recurrent cases 1 year or more after chemotherapy. During the follow-up period after chemotherapy, tissue with signal intensities similar to that of bone marrow was observed in a speckled pattern in the intramedullary space on T1- and T2-weighted images. This tissue was histologically demonstrated to be normal bone marrow and was considered to be bone marrow remaining after chemotherapy. In this small series, histological findings supported the results of MRI, confirming the usefulness of MRI for diagnosing bone marrow metastasis of neuroblastoma. However, bone marrow metastasis after chemotherapy was difficult to evaluate by comparing signal intensities alone. © 1993 Wiley-Liss, Inc.     

Detection of bone marrow involvement by neuroblastoma: Comparison of two cytological methods

In order to improve the assessment of bone marrow (BM) involvement by neuroblastoma, a study has been designated on aspirated material from ten BM sites. The classical method of smearing each BM aspirate (TK1) was compared to cytocentrifugation of the pool of BM samples after gradient density separation (TK2). Twenty smears from each technique were screened for the presence of tumor clumps. The screening was much easier and more rapid by TK2 than by TK1. Of the 103 procedures performed, 100 results were found concordant by both techniques, 3 were found negative by TK1 and positive by TK2. None was found positive by TK1 and negative by TK2. Of the 25 positive procedures, two had an equal number of positive smears by both techniques and 23 had more positive smears by TK2 than by TK1 (p < 0.001). Cytological examination of cytocentrifuge smears from the pool of BM samples after gradient density separation appears a simple, rapid and accurate technique for routine detection of BM involvement in neuroblastoma.     

Vacuolated cells in bone marrow aspirate of children with neuroblastoma

Cytoplasmic vacuoles that have been seen in FAB L3-type lymphoblasts are not usual in neuroblastoma. The authors report three children with neuroblastoma having vacuolated cells mimicking L3-type lymphoblasts on bone marrow aspiration smears.     

Immunocytochemical detection of neuroblastoma cells infiltrating clinical bone marrow samples

To evaluate the feasibility and clinical usefulness of immunocytochemical detection of bone marrow metastases in neuroblastoma, we studied bone marrow samples from patients undergoing intensive therapy, followed in the majority of cases by autologous bone marrow rescue. Two monoclonal antibodies were used in an indirect immunoenzymatic assay to test 384 samples collected from multiple bone marrow sites during 79 staging procedures in 48 patients. Of 578 immunocytochemical tests, 59 (10%) yielded non-evaluable results. Analysis by individual bone marrow sites showed an agreement between cytological and immunocytochemical examinations in 276 of 309 (89%) evaluable tests with 5 A7 and in 179 of 210 (85%) with UJ 13 A. Infiltration by neuroblastoma cells was reported in 9% of samples by cytology, in 6% by immunochemistry with 5 A7 and in 16% with 13 A. Analysis of results by staging demonstrated agreement between cytological examination and immunocytochemical detection with both monoclonal antibodies in 60 of 75 (80%) evaluable stagings. Bone marrow metastasis was detected by cytology in 22% of stagings, by immunochemistry with 5 A7 in 23%, with UJ 13 A in 25%. Detailed analysis of discordant results revealed that they were related partly to bone marrow sampling variability associated with focal and minimal metastasis of neuroblastoma cells. These data suggest the clinical usefulness of immunocytochemical detection as a complementary test to cytological examination for accurate evaluation of bone marrow infiltration in patients with disseminated neuroblastoma.Abbreviations IC immunocytochemical detection - BM bone marrow - Mabs monoclonal antibodies - CE cytological examination - NBL neuroblastoma - staging staging procedure Presented in part at the 19th meeting of the International Society of Paediatric Oncology, Jerusalem, 13–18 September 1987     

Cyto-morphologic evaluation of bone marrow in infants with disseminated neuroblastoma

We studied the prevalence and degree of tumor cell infiltration (TCI) in bone marrow (BM) aspirates of 89 infants with stage 4/4 S neuroblastoma and correlated them with MYCN gene status and patient outcome. TCI was scored 0, +, ++, and +++, the last corresponding to an infiltration greater than 10%. TCI 0 was more frequent in stage 4 than in stage 4 S. TCI + and ++ were equally represented. TCI +++ was found only in 9 patients, all with typical stage 4 features (bone or lung involvement). Overall survival was not significantly influenced by the presence and degree of TCI.     

Comprehensive treatment of advanced neuroblastoma involving autologous bone marrow transplant

Encouraging results are reported with high-dose chemotherapy and total body irradiation followed by autologous bone marrow transplantation in the treatment of advanced neuroblastoma. However, relapse remains a significant problem. We used high-dose chemotherapy, surgery, intraoperative radiation and an autologous bone marrow transplant treated in vitro to remove tumor cells followed by 13-cis-retinoic acid to treat 36 children with advanced neuroblastoma. This comprehensive treatment appears to improve the survival rate of patients with advanced neuroblastoma, including those with N-myc amplification and bony involvement. The disease-free survival rate was 66% (95% confidence interval, 49–84%) at 3 years. All patients who received 13-cis-retinoic acid developed cheilitis, but no bone marrow depression occurred in these patients. Five patients developed hemolytic uremic syndrome (HUS) post-transplant. This may have been related to the procedure used for total body irradiation. Patients who had their kidneys shielded during this procedure did not develop this syndrome. Patients who received local irradiation at the primary site showed no evidence of relapse in this region, indicating that such therapy may help to prevent a relapse. These data suggest a high rate of 3 year disease-free survival with this treatment strategy. The nonrandomized nature of the study and use of multiple modalities precludes analysis of the specific contribution of each.