Detection of micrometastasis of neuroblastoma to bone marrow and tumor dissemination to hematopoietic autografts using flow cytometry and reverse transcriptase‐polymerase chain reaction

Abstract

BACKGROUNDThe identification of neuroblastoma metastases to bone marrow (BM) is requisite in staging disease for risk‐adopted therapy. However, micrometastases were not elucidated fully.METHODSFlow cytometry (FCM) with CD45/CD56/CD81 and reverse transcriptase‐polymerase chain reactions (RT‐PCR) for tyrosine hydroxylase (TH) transcripts were used to evaluate neuroblastoma in bilateral BM aspirates at diagnosis, BM autografts, peripheral blood stem cell (PBSC) collections, and CD34+ cell products of 27 children.RESULTSTH transcripts were amplified in histology‐negative (H−) BM specimens from seven patients (four patients with Stage 3 disease, two with Stage 4 disease, and one with Stage 4S disease), revealing a prevalence of submicroscopic metastasis. The median number of CD45−CD81+CD56+ cells in four H− TH− BM samples from two patients with Stage 1 and Stage 2 disease, respectively, was comparable to that encountered in 10 normal BM controls (0.003% [range, 0.002–0.004%] vs. 0.004% [0–0.008%], P = 0.724). In six H− TH+ BM specimens from three patients whom were otherwise diagnosed with neuroblastoma Stage 3, 0.031% (0.009–0.06%) CD45−CD81+CD56+ cells were detected. Besides, 1.474% (0.088–3.009%) CD45−CD81+CD56+ cells were identified in four H− TH+ BM specimens from two patients at Stage 4. TH transcripts were evident in four of five BM autografts and in 22 of 45 (48.9%) PBSC specimens. FCM demonstrated 0.018% and 0.049% CD45−CD81+CD56+ cells in two TH+ BM autografts, respectively. The number of CD45−CD81+CD56+ cells was higher in 19 TH+ PBSC specimens than in 20 TH− PBSC specimens (0.026% [0.006–1.128%] vs. 0% [0–0.009%], P < 0.0001). CD34+ cell selection achieved 2.9 (2.1–3.5) log depletion of CD45−CD81+CD56+ cells in four manipulated products, rendering six of seven PBSC autografts TH‐free.CONCLUSIONSFCM in combination with RT‐PCR evaluated neuroblastoma micrometastasis and assessed the purity of hematopoietic autografts for transplant. However, the clinical relevance remains to be elucidated. Cancer 2003;97:2887–97. © 2003 American Cancer Society.DOI 10.1002/cncr.11389

Excellent Prognosis in a Subset of Patients with Ewing Sarcoma Identified at Diagnosis by CD56 Using Flow Cytometry

“…This method has also been used to identify circulating and/or micrometastatic tumor cells in adult patients with carcinoma and was proved to be a significant prognostic factor in patients with prostate cancer and breast carcinoma (11,12). In pediatric patients, micrometastatic cells were detected in neurobalstoma and rhabdomyosarcoma (13)(14)(15). Only a few reports used FC for the diagnosis of ES tumors for the combination of CD45À/CD99þ (22,27).…”

“…It is also used for diagnosis and follow-up evaluation in solid tumors, including breast and prostate cancer (11,12) in adult patients and rhabdomyosarcoma and neuroblastoma in pediatric patients (13)(14)(15).…”

Type III TGF-β receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

“…Expression of individual genes is often unreliable (47), and some in vitro differentiation markers, such as tyrosine hydroxylase and neuron-specific enolase, are markers of metastasis (48) and poor prognosis (49,50) when used clinically. In our meta-analysis of a microarray data set, none of the in vitro differentiation markers we used elsewhere correlated with stage of disease or TGFBR3 expression (data not shown).…”

Neuroblastoma Cells Isolated from Bone Marrow Metastases Contain a Naturally Enriched Tumor-Initiating Cell

“…The glycoprotein CD24 was investigated as a candidate unique identifier for the TIC in our neuroblastoma tumor spheres as this antigen has been shown to be expressed on renal cell carcinomas, small-cell lung carcinomas, and neuroblastomas (36,37). The hematopoietic progenitor marker CD34 was investigated as a potential unique identifier of the TIC in our neuroblastoma tumor spheres because several studies suggested that a small number of neuroblastoma cells in the bone marrow expressed CD34 and might be present in sufficient numbers in autologous bone marrow transplants to cause relapse (38)(39)(40)(41). We observed the presence of a small fraction of CD24 + (f0.24%) and CD34 + (f3.04%) cells in our highrisk neuroblastoma tumor spheres derived from bone marrow aspirates (Fig.…”