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

Abstract

The identification of neuroblastoma metastases to bone marrow (BM) is requisite in staging disease for risk-adopted therapy. However, micrometastases were not elucidated fully. Flow 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. TH 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. FCM 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.