Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level.

Abstract

In this report we describe a quantitative in vitro assay for the most primitive type of leukemic precursors yet defined in patients with chronic myeloid leukemia (CML). This assay is based on the recently described "long-term culture-initiating cell" (LTC-IC) assay for primitive normal human hematopoietic cells. Such cells, when cocultured with competent fibroblast feeder layers, give rise after a minimum of 5 weeks to multiple single and multilineage clonogenic progenitors detectable in secondary semisolid assay cultures. Similar cultures initiated by seeding a highly enriched source of leukemic cells from patients onto normal feeders showed the clonogenic cell output after 5 weeks to be linearly related to the input innoculum over a wide range down to limiting numbers of input cells, thus allowing absolute frequencies of leukemic LTC-ICs to be determined using standard limiting dilution analysis techniques. Leukemic LTC-IC concentrations in CML marrow were found to be decreased, on average to <10% ofthe normal LTC-IC concentration in normal marrow, but were greatly increased (up to > 10 times) in CML blood. Assessment of the number of clonogenic cells produced per leukemic LTC-IC by comparison to normal blood or marrow LTC-IC values showed this function to be unchanged in leukemic LTC-ICs [i.e., 3.1 ± 0.4 clonogenic cells per CML LTC-IC (mean ± SEM, n = 6) versus 3.7 ± 1.2 (n = 3) and 4.3 ± 0.4 (n = 5), respectively, for normal blood and marrow LTC-ICs].In contrast, leukemic LTC-IC maintenance in LTC proved to be highly defective by comparison to normal LTC-IC of either blood or marrow origin. Thus, when cells from primary LTC were subcultured into secondary LTC-IC assays, leukemic LTC-IC rapidly declined (>30-fold) within the first 10 days of culture, whereas normal LTC-IC numbers remained unchanged during this period. These findings illustrate how self-maintenance and differentiation events in primitive human hematopoietic cells can be differentially modulated by an oncogenic process and provide a framework for further studies of their manipulation, analysis, and therapeutic exploitation. (Ph') (1). The initial cell transformed, and hence the origin of the leukemic clone, is believed to be a totipotent hematopoietic cell with lymphoid as well as myeloid differentiation potential since Ph'-positive cells in these lineages are frequently demonstrable (2). This has suggested that production of the BCR-ABL kinase in a totipotent hematopoietic cell gives it a selective growth advantage. Recent experiments involving retroviral infection of murine bone marrow (BM) cells with BCR-ABL constructs are consistent with this (3, 4), although an underlying molecular mechanism has not been determined. In particular, the biological consequences of BCR-ABL kinase expression in very primitive human hematopoietic cells have been difficult to investigate because methods for their selective isolation have not been available.CML patients with elevated leukocyte (WBC) counts show dramatic increases in the number of Ph'-posi...

BCR/ABL-negative progenitors are enriched in the adherent fraction of CD34+ cells circulating in the blood of chronic phase chronic myeloid leukemia patients

“…Opinions range from the one extreme, that the great recently been used by Maguer-Satta et al 29 They studied five majority of primitive cells are Ph − , to the other, that many CML patients in whom the primitive cell compartment (LTCIC) are Ph + at least in some patients. 11,[13][14][15][16][17]29,30 However, recent was known to consist predominantly of Ph + cells. By sorting studies demonstrating the existence in CML patients of leuprimitive cells and performing RT-PCR on them individually kemic cells that are capable of reconstituting severe combined they found that BCR/ABL mRNA could be specifically immunodeficient mice, 31 and the well-demonstrated transdetected in about 60-90% of the CML cells in which the actin forming activity of BCR-ABL in experimental systems, [32][33][34] message was present.…”

Allografting in chronic myeloid leukemia with cultured marrow: Update of the vancouver study

“…Nine of these twelve patients were then treated with a-interferon (IFN-a) 1-3 x lo6 units/m2, 3-7 dayslweek; four returned to complete remission, three developed increasing numbers of Ph+ cells, and two are still too early to evaluate. Recently, we have shown that leukemic stem cells, operationally defined as Ph+ LTC initiating cells (LTC-IC), are, on average, present in CML marrow at levels 10-fold lower than their normal counterparts and can be selectively "purged" (by a factor of 30-fold) following incubation for 10 days in LTC [5,6].…”

Biological strategies for the selective manipulation of normal and leukemic stem cells

“…Initially, the finding that such cells were present in markedly elevated numbers in the peripheral blood, as anticipated from previous studies of clonogenic cells, led to the expectation that their numbers might be similarly elevated in the marrow. In fact, the opposite appears to be the case [19] (see also Fig. IB).…”

“…The most dramatic differential effect of any treatment thus far examined appears to be that afforded by simply placing a CML marrow autograft in culture for a period of 7 to 10 days [19]. Under these conditions, normal LTC-IC numbers remain at, or may even increase to slightly above, input numbers whereas the Ph-positive LTC-IC decline precipitously, on average by a factor of -30 (Fig.…”