Purging of human breast cancer cells from stem cell products with an adenovirus containing p53

Abstract

Tumor cell contamination of stem cell products can contribute to tumor relapse following high-dose chemotherapy and stem cell rescue. Numerous techniques have been used to remove the tumor cells from stem cell products with the objective of prolonging relapse-free survival. However, to date these techniques have been relatively ineffectual and/or toxic to hematopoietic stem and progenitor cells. The differential infectivity of adenovirus (Adv) vectors for breast cancer cells, compared with hematopoietic cells, has suggested that Adv-p53 might provide an effective purging strategy. To facilitate the use of Adv-p53 as a clinical strategy, we undertook studies to determine the parameters necessary for optimal stem cell product purging. The parameters studied were the particle number to nucleated cell ratio, the duration of coincubation, the incubation volume, and the presence or absence of hematopoietic progenitor cells. We have found that these parameters are interdependent and conclude that a 4-hour coincubation with an Adv-p53 particle to nucleated cell ratio of 2000:1 with 2 ϫ 10 8 nucleated cells/mL is optimal for tumor cell purging. Furthermore, this appeared to be a safe procedure, with total loss of clonogenic growth of breast cancer cells as well as no significant effect on progenitor cell function as determined by granulocyte-macrophage colony-forming unit assays. Cancer Gene Therapy ( T reatment with high-dose chemotherapy (HDT) followed by stem cell rescue is used increasingly in patients with breast cancer. 1-5 However, a high relapse rate after HDT is still observed, especially in women with high risk and metastatic breast cancer. 6 In most cases, failure of the conditioning regimen to eradicate disease remains the primary cause of disease recurrence. There is strong evidence from gene-marking studies that tumor cell (TC) contamination of the graft can also contribute to cancer recurrence, 7 although such studies have not been undertaken with breast cancer transplant patients. 8,9 TC can be detected not only in the bone marrow (BM) of advanced breast cancer patients (20 -70%) but also in the BM of patients with localized breast cancer (20 -45%) using sensitive immunocytochemical analyses 9 or reverse transcriptase-polymerase chain reaction assays. 10 -14 Mobilized peripheral blood stem cell (PSC) products are used as an alternative to BM stem cells and result in a more rapid neutrophil recovery 3,5 following HDT. Initially, it appeared that PSC products were free of TC; however, recent studies have demonstrated that cancer cells can also be detected in mobilized PSC products. 15,16 Although PSC products have a reduced level of TC contamination compared with BM cells, 5,15,17,18 they are frequently contaminated with TC, and novel strategies for PSC purging are needed.Clinically, mutations of wild-type (wt) p53 are observed in 30 -50% of breast cancer patients and functional inactivation of p53 may occur in 30% of these individuals. 19 Transduction of wt p53 into TC with mutated p53 can suppress the ma...

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34+ cells

“…17,22,23 This selectivity may be uniquely exploited in the purging of tumor cells from stem cell products and we have previously shown that the conditions used for these studies can result in six -log tumor cell killing by rAdp53. 18 This finding suggests that an rAd -p53 tumor cell purging protocol may provide a two-to four-log increase in efficacy when compared to conventional strategies. 25 Another advantage to the use of an rAd -p53 tumor cell purging strategy includes the absence of additional steps following transplantation.…”

“…Our protocol was designed to minimize handling of the stem cell products and used a co -incubation with rAd -p53 for a period of 4 hours at 378C. 18 The concentration of vector selected ( 2Â10 11 rAd -p53 particles / mL ) was previously determined to provide a six -log tumor cell reduction in cytokine -mobilized stem cell products. 18 The studies presented here have enabled us to quantify the effect of co -culturing isolated, cytokine -mobilized CD34 + cells with clinically relevant concentrations of rAd -p53 and to determine the effect of this treatment on subsequent engraftment and multilineage hematopoietic reconstitution of NOD -SCID recipients.…”

“…18 The concentration of vector selected ( 2Â10 11 rAd -p53 particles / mL ) was previously determined to provide a six -log tumor cell reduction in cytokine -mobilized stem cell products. 18 The studies presented here have enabled us to quantify the effect of co -culturing isolated, cytokine -mobilized CD34 + cells with clinically relevant concentrations of rAd -p53 and to determine the effect of this treatment on subsequent engraftment and multilineage hematopoietic reconstitution of NOD -SCID recipients. Here we provide evidence that SRA, multipotency, and self -renewal of stem cell products are not compromised by co -incubation with rAd -p53 at targeted clinical levels.…”

“…13,14,24 The apparent absence of detrimental effects of co -incubation of stem cell products with rAd -p53 as a purging strategy reported here is an encouraging finding and consistent with our previous experience. 17,18,27,28 In contrast to the studies with flow cytometry and PCR using an LDA, studies examining the formation of CFU -GM were uninformative. In all the groups of mice, whether receiving rAd -p53 co-incubated or control CD34 + cells, a significant increase in the frequency of CFU -GM was observed relative to the mice receiving lethally irradiated human carrier cells.…”

“…17 Indeed, the co-incubation of tumor cell -contaminated stem cell products with rAd -p53 reduced tumor cell numbers by six logs. 18,24 The efficacy of this strategy is underscored by the observation that in addition to preferential transduction, the target replacement gene, p53, is mutated in over 50% of breast cancer patients Whereas previous studies have shown that the coincubation of stem cell products with rAd -p53 does not impact in vitro hematopoietic progenitor cell function, 27,28 these findings do not address the sequelae of rAd -p53 co -incubation on the primitive HSC compartment critical for successful HSC transplantation. A murine model [ nonobese diabetic /severe combined immunodeficient ( NOD -SCID ) mouse] , which allows such studies, 29,30 has been developed.…”