Effectiveness of telehealth in preventive care: a study protocol for a randomised controlled trial of tele-exercise programme involving older people with possible sarcopenia or at risk of fall.

BACKGROUND: Continuous loss of muscle mass and strength are the consequences of the ageing process, which increase the risk of falls among older people. Falls can lead to severe consequences such as bone fractures and hampered physical and psychological well-being. Regular exercise is the key to reversing muscle atrophy and relieving sarcopenia. However, the frailty of older people and the recent COVID-19 pandemic may affect their confidence to leave home to attend classes in the community. A feasible and effective alternative should be explored. METHODS: The primary objective is to evaluate the effectiveness of tele-exercise (TE) in relation to physical functioning and exercise adherence among community-dwelling older people at risk of falls in comparison with a community-based group (CB). The secondary objective includes evaluating older people's experience with tele-exercise, emphasizing their psychological welfare, social well-being, and acceptance of the telehealth approach. The design, conduct, and report follow the SPIRIT guidelines (Standard Protocol Items: recommended items to address in a Clinical Trial Protocol and Related Documents). Older people will be recruited from 10 local community centres in Hong Kong and randomly allocated into two groups. All participants will attend the exercise training 3 days per week for 3 months but the mode of delivery will differ, either online as the tele-exercise group (TE) or face-to-face as the community-based group (CB). The outcome measures include muscle strength, physical function, exercise adherence and dropout rate, psychological and social well-being will be assessed at the baseline, and the 3rd, 6th and 12th month. Some participants will be invited to attend focus group interviews to evaluate their overall experience of the tele-exercise training. DISCUSSION: Tele-exercise reduces the barriers to exercise, such as time constraints, inaccessibility to facilities, and the fear of frail older people leaving their homes. Promoting an online home-based exercise programme for older people can encourage them to engage in regular physical activity and increase their exercise adherence even when remaining at home. The use of telehealth can potentially result in savings in cost and time. The final findings will provide insights on delivering exercise via telehealth to older people and propose an exercise delivery and maintenance model for future practice. TRIAL REGISTRATION: Chinese Clinical Trial Registry ( https://www.chictr.org.cn/hvshowprojectEN.html?id=219002&v=1.1 ), registration number: ChiCTR2200063370. Registered on 5 September 2022.

Outcomes of Stent Retriever versus Aspiration-First Thrombectomy in Ischemic Stroke: A Systematic Review and Meta-Analysis.

BACKGROUND: There is ongoing debate regarding the optimal first-line thrombectomy technique for large-vessel occlusion. PURPOSE: We performed a systematic review and meta-analysis of comparative studies on stent retriever-first and aspiration-first thrombectomy. DATA SOURCES: We searched Ovid MEDLINE, PubMed, and EMBASE from 2009 to February 2018. STUDY SELECTION: Two reviewers independently selected the studies. The primary end point was successful reperfusion (TICI 2b/3). DATA ANALYSIS: Random-effects meta-analysis was used for analysis. DATA SYNTHESIS: Eighteen studies including 2893 patients were included. There was no significant difference in the rate of final successful reperfusion (83.9% versus 83.3%; OR = 0.87; 95% CI, 0.62%-1.27%) or good functional outcome (mRS 0-2) at 90 days (OR = 1.07; 95% CI, 0.80-1.44) between the stent-retriever thrombectomy and aspiration groups. The stent-retriever thrombectomy-first group achieved a statistically significant higher TICI 2b/3 rate after the first-line device than the aspiration-first group (74.9% versus 66.4%; OR = 1.53; 95% CI, 1.14%-2.05%) and resulted in lower use of a rescue device (19.9% versus 32.5%; OR = 0.36; 95% CI, 0.14%-0.90%). The aspiration-first approach resulted in a statistically shorter groin-to-reperfusion time (weighted mean difference, 7.15 minutes; 95% CI, 1.63-12.67 minutes). There was no difference in the number of passes, symptomatic intracerebral hemorrhage, vessel dissection or perforation, and mortality between groups. LIMITATIONS: Most of the included studies were nonrandomized. There was significant heterogeneity in some of the outcome variables. CONCLUSIONS: Stent-retriever thrombectomy-first and aspiration-first thrombectomy were associated with comparable final reperfusion rates and functional outcome. Stent-retriever thrombectomy was superior in achieving reperfusion as a stand-alone first-line technique, with lower use of rescue devices but a longer groin-to-reperfusion time.

Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force.

Disseminating disease is a predictive and prognostic indicator of poor outcome in children with neuroblastoma. Its accurate and sensitive assessment can facilitate optimal treatment decisions. The International Neuroblastoma Risk Group (INRG) Task Force has defined standardised methods for the determination of minimal disease (MD) by immunocytology (IC) and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) using disialoganglioside G(D2) and tyrosine hydroxylase mRNA respectively. The INRG standard operating procedures (SOPs) define methods for collecting, processing and evaluating bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell harvest by IC and QRT-PCR. Sampling PB and BM is recommended at diagnosis, before and after myeloablative therapy and at the end of treatment. Peripheral blood stem cell products should be analysed at the time of harvest. Performing MD detection according to INRG SOPs will enable laboratories throughout the world to compare their results and thus facilitate quality-controlled multi-centre prospective trials to assess the clinical significance of MD and minimal residual disease in heterogeneous patient groups.

Expression of SSX-2 and SSX-4 genes in neuroblastoma.

The SSX genes are members of the family of cancer/testis antigens that encode tumor-associated antigens recognizable by autologous cytolytic T lymphocytes. Their expression is common in tumors of diverse lineages and absent in normal tissues except testis and thyroid. In this study, sixty-seven neuroblastomas (NB) (12 stage 1, 13 stage 2, 12 stage 3, 12 stage 4S and 13 stage 4) were examined by RT-PCR and a sensitive chemiluminescent detection method for SSX-2 and SSX-4 expression. Seventy-two percent (13/18) of stage 4 NB expressed SSX-2 and 67% (12/18) expressed SSX-4. SSX-2 and SSX-4 positivity correlated with metastatic NB stage 4 (p=0.02 and p=0.006, respectively). Sensitivity experiments showed SSX-2 detection was one tumor cell in 10(6) normal cells, and one in 10(4) for SSX-4. All normal tissues (n=6), with the exception of testis, normal bone marrow (BM, n=12) and normal peripheral blood (PBL, n=10) were negative for SSX-2 and SSX-4 expression. Thirty-two BM and 14 PBL obtained from 35 stage 4 NB patients at 24 months from their diagnosis were evaluated for SSX-2 expression. Unlike another cancer/testis antigen, GAGE, only one BM sample was positive, and no prognostic utility could be established. Further investigation of SSX expression at other relevant time points is warranted.

GD2 synthase: a new molecular marker for detecting neuroblastoma.

BACKGROUND: Neuroblastomas (NBs) almost ubiquitously express the ganglioside GD2. GD2 synthesis is dependent on the key enzyme GD2 synthase. Thus, GD2 synthase transcript may prove to be a potential molecular marker of NB. METHODS: Seventy-seven NB tumor tissues of all stages, 5 NB cell lines, and 26 normal bone marrows (BMs) and peripheral blood (PBL) samples, as well as 26 non-NB remission-BMs were analyzed for the expression of GD2 synthase by a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) and chemiluminescence detection. One hundred fifty-two NB BMs were tested and comparisons were made among three independent detection techniques, namely GD2 synthase RT-PCR, immunofluorescence (IF), and histology (HIST). RESULTS: GD2 synthase transcript was present in 5 of 5 cell lines and in 77 of 77 tumors tested. Among 116 marrows that were positive by at least 1 of the 3 methods, 78% were detectable by GD2 synthase, 68% by IF, and 46% by HIST. Seventy-six percent of positive BMs that were obtained during treatment and follow-up had GD2 synthase expression, whereas only 29% were HIST positive. Correlation between RT-PCR and IF was high (P = 0.001), and positivity by 3 out of 3 methods was strongly correlated with poor survival (P < 0.01). Of note, marrows tested at the time of chemotherapy were positive by at least 2 out of 3 methods and were associated with adverse outcome (P = 0.01). Serial samples (n = 28) in 5 patients demonstrated close agreement between RT-PCR and patient disease status. CONCLUSIONS: The current study found that molecular detection of GD2 synthase transcript in NB BMs may have potential value in detecting rare tumor cells.

Neuroblastic and Schwannian stromal cells of neuroblastoma are derived from a tumoral progenitor cell.

The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages.

Hox D3 expression in normal and impaired wound healing.

BACKGROUND: We have previously shown that Hox D3 and Hox B3 can promote angiogenesis. As angiogenesis is essential for wound healing, we examined expression of these genes in the vasculature following wounding in normal and genetically diabetic adult mice with impaired healing. METHODS: In situ hybridization was performed on tissues taken 0, 1, 4, 7, and 14 days following administration of linear wounds in wild-type and genetically diabetic mice. Expression of Hox D3 and Hox B3, angiogenesis, and synthesis of type I collagen were assessed in the wound. RESULTS: Hox B3 was expressed in endothelial cells (ECs) of both medium and small vessels in unwounded tissue, whereas little Hox D3 was detected in resting ECs. Hox D3 expression was significantly upregulated by 1 day after wounding in ECs of vessels immediately adjacent to the wound site, and expression was maintained for at least 7 days. In the diabetic mice, expression of Hox B3 was similar to that of wild-type mice. In contrast, expression of Hox D3 in ECs was significantly lower and delayed during wound repair in diabetic mice. In cultured microvascular ECs, Hox D3 selectively induced high levels of collagen I mRNA expression. Hox D3-deficient wounds of diabetic animals also displayed a reduction in expression and deposition of type I collagen. CONCLUSIONS: These results suggest that reduced angiogenesis and type I collagen in diabetic mice with impaired wound healing may be related to deficient Hox D3 expression, and restoring Hox D3 expression may enhance angiogenesis and wound repair.

Detection of microscopic disease: comparing histology, immunocytology, and RT-PCR of tyrosine hydroxylase, GAGE, and MAGE.

BACKGROUND: We first explored the use of multiple molecular markers to overcome tumor heterogeneity. Sixty-seven neuroblastoma (NB) tumors were tested for the expression of GAGE, MAGE-2, MAGE-2, MAGE-3, and MAGE-4 by RT-PCR and then chemiluminescence; 82% of tumors had detectable GAGE, and 88% expressed at least one of the four MAGE genes. PROCEDURE AND RESULTS: By combining GAGE and MAGE, 64 of 67 (95%) of tumors became detectable; 17 of 67 coexpressed all five molecular markers. Neither GAGE nor MAGE expression correlated with stage. GAGE was found to have the broadest (18 of 18) expression among stage 4 tumors. Two hundred fifty-nine bone marrows from 99 patients were then studied for NB positivity by four detection methods: histology, immunocytology, and molecular detection by GAGE and tyrosine hydroxylase (TH) mRNA. Two hundred seven samples were NB-positive by one detection method. All four techniques were comparable in detecting tumor cells at diagnosis and at relapse. GAGE and immunocytology were far more sensitive than histology and TH mRNA when marrows were sampled during chemotherapy and at the time of clinical remission. CONCLUSIONS: By combining multiple molecular markers and independent screening techniques, we may be able to overcome tumor heterogeneity and expedite the detection of microscopic disease in the clinical management of neuroblastoma.

N7: a novel multi-modality therapy of high risk neuroblastoma (NB) in children diagnosed over 1 year of age.

BACKGROUND: The N7 protocol for poor-risk neuroblastoma uses dose-intensive chemotherapy (as in N6 protocol [Kushner et al.: J Clin Oncol 12:2607-2613, 1994] but with lower dosing of vincristine) for induction, surgical resection and 2100 cGy hyperfractionated radiotherapy for local control, and for consolidation, targeted radioimmunotherapy with 131I-labeled anti-GD2 3F8 monoclonal antibody and immunotherapy with unlabeled/unmodified 3F8 (400 mg/m2). PROCEDURE: The chemotherapy consists of: cyclophosphamide 70 mg/kg/d x 2 and a 72-hr infusion of doxorubicin 75 mg/m2 plus vincristine 2 mg/m2, for courses 1, 2, 4, and 6; and cisplatin 50 mg/m2/d x 4 and etoposide 200 mg/m2/d x 3, for courses 3, 5, and 7. 131I-3F8 is dosed at 20 mCi/kg, which is myeloablative and therefore necessitates stem-cell support. RESULTS: Of the first 24 consecutive previously untreated patients more than 1 year old at diagnosis, 22 were stage 4 and two were unresectable stage 3 with MYCN amplification. Chemotherapy achieved CR/VGPR in 21 of 24 patients. Twenty patients to date have completed treatment with 131I-3F8, and 15 patients have completed all treatment. With a median follow-up of 19 months, 18 of 24 patients remain progression-free. CONCLUSIONS: Major toxicities were grade 4 myelosuppression and mucositis during chemotherapy, and self-limited pain and urticaria during antibody treatment. Late effects include hearing deficits and hypothyroidism.

Quantitation of marrow disease in neuroblastoma by real-time reverse transcription-PCR.

PURPOSE: GD2 is abundantly expressed in neuroblastoma (NB). GD2 synthesis is dependent on key enzyme beta 1,4-N-acetylgalactosaminyltransferase (GD2 synthase). We explore the potential of GD2 synthase mRNA as a molecular marker of minimal residual disease by first comparing it quantitatively with immunocytology and then testing its clinical utility. EXPERIMENTAL DESIGN: A real-time reverse transcription-PCR assay to quantify mRNA of GD2 synthase was developed. Quantitation was normalized to endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex PCR. RESULTS: The upper limit of normal was defined by 31 normal marrow and blood samples, achieving a sensitivity of one NB cell in 10(6) normal mononuclear cells. When 155 bone marrows from 100 NB patients were studied, GD2 synthase mRNA levels correlated well with the number of GD2-positive cells, as measured by immunocytology using anti-GD2 antibodies (r = 0.96). This is the first demonstration of the quantitative relationship between a specific mRNA and the actual number of tumor cells. In a pilot study, the level of this transcript in sequential marrow samples of five stage 4 NB patients correlated closely with their clinical status. At 24 months after diagnosis, available remission bone marrows from patients with advanced NB diagnosed at >1 year of age initially treated with protocols N6 and N7 at Memorial Sloan-Kettering Cancer Center (n = 44) were analyzed for GD2 synthase mRNA. Positivity was strongly associated with progression-free (P < 0.005) and overall survival (P < 0.001). CONCLUSIONS: Measurement of tumor cells by real-time quantitative reverse transcription-PCR of GD2 synthase has potential clinical utility, especially for the detection of minimal residual disease.

Prognostic significance of GAGE detection in bone marrows on survival of patients with metastatic neuroblastoma.

BACKGROUND: Previously, we reported the utility of GAGE as a molecular marker for neuroblastoma (NB) and malignant melanoma in blood and bone marrow (BM). Among patients with stage III melanoma rendered disease-free by surgery, GAGE expression was a strong prognostic factor for patient survival. PROCEDURE: All patients with advanced NB diagnosed at > 1 year of age initially treated with protocol N6 (n = 24) and N7 (n = 38) at Memorial Sloan-Kettering Cancer Center were included in this study. Their BM cells at 12, 18, and 24 months (median time after diagnosis) were evaluated for the presence of GAGE. RESULTS: GAGE positivity at 12 months (25%), when patients were still on treatment, did not predict progression-free survival (PFS) and overall survival from the time of sampling. Positivity at 18 months (29%) was associated with poorer PFS and survival (but P > 0.05). By 24 months, the presence of GAGE (26%) was a very strong predictor of out-come (P < 0.001). When only remission marrows at 24 months were analyzed, PFS was 4.7-fold lower among GAGE-positive patients. Thirty-seven percent of N6 patients were positive for GAGE, in contrast to 17% of the patients in the more current regimen N7. CONCLUSIONS: The detection of GAGE by RT-PCR in marrow may have utility in molecular staging of patients in clinical remission. It may allow earlier identification of patients at risk, such that appropriate intervention can be given before clinical relapse. GAGE may also serve as a surrogate endpoint for adjuvant treatment strategies, and to determine the duration of therapy.

Correlation of anti-idiotype network with survival following anti-G(D2) monoclonal antibody 3F8 therapy of stage 4 neuroblastoma.

BACKGROUND: A transient human anti-mouse antibody response was associated with significantly longer survival [Cheung et al. (1998): J Clin Oncol 16:3053] following antibody 3F8 (Ab1) treatment. We postulate that the induction of an idiotype network which included anti-anti-idiotypic (Ab3) and anti-G(D2) (Ab3') responses is associated with tumor control. PROCEDURE: Thirty-four patients with stage 4 neuroblastoma (NB) diagnosed at > 1 year of age were treated with anti-G(D2) monoclonal antibody 3F8 at the end of chemotherapy RESULTS: Long-term progression-free survival and overall survival correlated significantly with Ab3' andAb3, but not with non-idiotypic antibody responses. Only one of six individual specificities showed significant correlations with patient survival. CONCLUSIONS: As in vitro correlates of idiotype network initiated by Ab1 treatment, Ab3 and Ab3' may provide convenient biologic endpoints for monoclonal antibody therapy of advanced NB, and a rationale for choosing specific anti-idiotypic antibodies for vaccine development.

Induction of Ab3 and Ab3' antibody was associated with long-term survival after anti-G(D2) antibody therapy of stage 4 neuroblastoma.

Treatment with anti-G(D2) monoclonal antibody 3F8 (Ab1) at the time of remission may prolong survival for children with stage 4 neuroblastoma. A transient human antimouse antibody (HAMA) response was associated with significantly longer survival (Cheung et al., J. Clin. Oncol., 16: 3053-3060, 1998). Because this response was primarily anti-idiotypic (Ab2), we postulate that the subsequent induction of an idiotype network that included an elevation of anti-anti-idiotypic (Ab3) and anti-G(D2) (Ab3') antibody titers may be responsible for tumor control. Thirty-four patients with stage 4 neuroblastoma diagnosed at >1 year of age were treated with 3F8 at the end of chemotherapy. Most had either bone marrow (31 of 34) or distant bony (29 of 34) metastases at diagnosis. Thirteen patients were treated at second or subsequent remission, and 12 patients in this group had a history of progressive/persistent disease after bone marrow transplantation; 21 patients were treated in the first remission after N6 chemotherapy. Their serum HAMA, Ab3, and Ab3' titers prior to, at 6, and at 14 months after antibody treatment were measured by ELISA. Among these 34 patients, 14 are alive, and 13 (1.8-7.4 years at diagnosis) are progression free (53-143 months from the initiation of 3F8 treatment) without further systemic therapy. Long-term progression-free survival (PFS) and survival correlated significantly with Ab3' (anti-G(D2)) response at 6 months and with Ab3 response at 6 and 14 months. By defining Ab3 threshold ranging from the ratio of 1.1 to 2.6 above pretreatment level, the difference in PFS and survival between the high-Ab3 and low-Ab3 groups became markedly widened. Similarly, increasing the Ab3' threshold at either 6 or 14 months to 300% above pre-3F8 levels also increased the spread between the high versus low Ab3' groups for both PFS and survival curves. Non-idiotype antibody responses (anti-mouse-IgG3 or anti-tumor nuclear HUD antigen) had no apparent impact on PFS or survival. In conclusion, despite the high-risk nature of stage 4 neuroblastoma, long-term remission without myeloablative therapy can be achieved with 3F8 treatment. Ab3 and Ab3' antibody response correlated with prolonged PFS and survival. We postulate that successful induction of an idiotype network in patients may be responsible for long-term tumor control.

A method for detecting abasic sites in living cells: age-dependent changes in base excision repair.

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP-DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA-HRP complex at each biotin residue, which is rapidly separated from free avidin-HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.

Association between molecular detection of GAGE and survival in patients with malignant melanoma: a retrospective cohort study.

We used GAGE as a molecular marker to identify melanoma cells with metastatic potential in the peripheral blood and the bone marrow. One hundred thirty-three patients with malignant melanoma (21 clinical stage II, 74 stage III, and 38 stage IV) had a single marrow and/or blood sample drawn immediately prior to surgical resection. Simultaneous bone marrow and blood samples (85 patients), marrow-only samples (35 patients), and blood-only samples (13 patients) were examined for the presence of GAGE expression using reverse transcription-PCR. GAGE expression was associated with adverse overall patient survival, measured from the time of sampling (P = 0.01). When data were stratified for clinical stage, median survival was statistically longer among GAGE-negative patients in the stage III cohort only (P = 0.01). In a multivariate model, only GAGE positivity in blood and/or marrow and clinical stage were significant prognostic variables. It was the detection of GAGE in blood but not marrow that was associated with poor survival. The detection of blood GAGE by reverse transcription-PCR has significant adverse implications for overall survival of patients with malignant melanoma in this cohort, and it warrants further investigation.

The modulation of skeletal muscle contraction by FMRFamide-related peptides of the locust.

The external ventral protractor muscle of the VIIth abdominal segment, M234, is a skeletal muscle that possesses receptors that recognize a range of FMRFamide-related peptides and application of these peptides results in an increase in the amplitude of neurally evoked contractions with little or no effect on basal tonus. FLRFamide itself has the same biologic activity as the extended peptides, whereas truncation to LRFamide or RFamide results in a peptide with no biologic activity. The receptors recognizing these extended FLRFamides, which include SchistoFLRFamide, seem to be different from the SchistoFLRFamide receptors found on locust oviduct visceral muscle. SchistoFLRFamide and the non-peptide mimetic, benzethonium chloride (Bztc), increase the frequency and amplitude of miniature endplate potentials, increase the amplitude of neurally evoked excitatory junction potentials, and result in a hyperpolarisation of resting membrane potential. Bztc, however, also abolishes the active membrane response that may explain its ability to decrease neurally evoked contractions.

Detection of microscopic neuroblastoma in marrow by histology, immunocytology, and reverse transcription-PCR of multiple molecular markers.

We explored the use of multiple molecular markers to overcome tumor heterogeneity. Sixty-seven neuroblastoma (NB) tumors were tested for the expression of GAGE, MAGE-1, MAGE-2, MAGE-3, and MAGE-4 by reverse transcription-PCR. Eighty-two percent of 67 NB tumors had detectable GAGE, and 88% expressed at least one of the four MAGE genes. By combining GAGE and MAGE, we found that 64 of 67 (95%) of tumors became detectable and 17 of 67 coexpressed all five molecular markers. Neither GAGE nor MAGE expression correlated with stage. GAGE was found to have the broadest (18 of 18) expression among stage 4 tumors. A total of 259 bone marrows from 99 patients were then studied for NB positivity by four detection methods: histology (aspirate by Wright-Giemsa and biopsy by H&E staining), immunocytology (by a panel of anti-GD2 monoclonal antibodies), and molecular detection by GAGE and tyrosine hydroxylase mRNA. Two hundred seven samples were NB positive by one or more detection methods. All four techniques were comparable in detecting tumor cells at diagnosis. GAGE and immunocytology were more sensitive than histology or tyrosine hydroxylase reverse transcription-PCR when marrows were obtained from patients on therapy or off therapy during clinical remission. Agreement among tests was highest at the time of gross disease. We conclude that, by combining multiple molecular markers and independent screening techniques, we may be able to overcome tumor heterogeneity and expedite the detection of microscopic disease in the clinical management of NB.

Anti-G(D2) antibody treatment of minimal residual stage 4 neuroblastoma diagnosed at more than 1 year of age.

PURPOSE: To eradicate minimal residual disease with anti-G(D2) monoclonal antibody 3F8 in stage 4 neuroblastoma (NB) diagnosed at more than 1 year of age. PATIENTS AND METHODS: Thirty-four patients were treated with 3F8 at the end of chemotherapy. Most had either bone marrow (n=31) or distant bony metastases (n=29). Thirteen patients were treated at second or subsequent remission (group I) and 12 patients in this group had a history of progressive/persistent disease after bone marrow transplantation (BMT); 21 patients were treated in first remission following N6 chemotherapy (group II). RESULTS: Before 3F8 treatment, 23 patients were in complete remission CR, eight in very good partial remission (VGPR), one in partial remission (PR), and two had microscopic foci in marrow. Twenty-five had evidence of NB by at least one measurement of occult/minimal tumor (iodine 131[(131)I]-3F8 imaging, marrow immunocytology, or marrow reverse-transcriptase polymerase chain reaction [RT-PCR]). Acute self-limited toxicities of 3F8 treatment were severe pain, fever, urticaria, and reversible decreases in blood counts and serum complement levels. There was evidence of response by immunocytology (six of nine), by GAGE RT-PCR (seven of 12), and by (131)I-3F8 scans (six of six). Fourteen patients are alive and 13 (age 1.8 to 7.4 years at diagnosis) are progression-free (40 to 130 months from the initiation of 3F8 treatment) without further systemic therapy, none with late neurologic complications. A transient anti-mouse response or the completion of four 3F8 cycles was associated with significantly better survival. CONCLUSION: Despite high-risk nature of stage 4 NB, long-term remission without autologous (A)BMT can be achieved with 3F8 treatment. Its side effects were short-lived and manageable. The potential benefits of 3F8 in consolidating remission warrant further investigations.

Detection of metastatic neuroblastoma in bone marrow: when is routine marrow histology insensitive?

PURPOSE: To measure the sensitivity of histologic examination in detecting metastatic solid tumor in bone marrow. PATIENTS AND METHODS: A total of 145 patients with stage 4 neuroblastoma underwent 840 marrow examinations, each consisting of six sites (four aspirates and two biopsies), from October 1990 to June 1996 at Memorial Sloan-Kettering Cancer Center. Metastasis was detected by either histology (aspirate by Wright-Giemse and biopsy by Hematoxylin-Eosin stains) or immunostaining of aspirates using anti-G(D2) monoclonal antibodies. RESULTS: The absence of tumor by histology at a single marrow site was a poor guarantee of the absence of disease. The number of false-negative sites increased as the percent of G(D2)-positive tumor cells in the marrow decreased: zero of six if tumor cell count was > or = 1%, and approximately six of six sites if < or = 0.003%. Sensitivity was comparable between marrow aspirate and biopsy. A lower bound (LB) for the probability of false-negative histology was calculated from the (1) discordance among the six marrow samplings and (2) comparison with immunofluorescence. When disease was extensive (eg, at diagnosis), the LB was 0.13 and 0.3, respectively. After treatment, it increased to 0.37 and 0.8. Examining multiple marrow sites can decrease the LB to 0.15. However, at least three sites have to be negative at relapse, six at diagnosis, and more than 50 during treatment or off-therapy follow-up. The marginal decrease in the LB by additional samplings rapidly diminished to less than 0.05 after two sites. CONCLUSION: Except at diagnosis and relapse when gross disease is present, marrow sampling by histology has limited sensitivity. Current practice grossly underestimates the true prevalence of marrow disease.