Contamination of leukapheresis products by tumor cells might be responsible for disease recurrence after autologous PBSC transplant in patients with solid tumors (Brenner, 1993), and different purging strategies are exploited to reduce the tumor cell content of PBSC grafts. Neoplastic cells have been shown to be more sensitive than normal hemopoietic stem cells to freezing techniques (Allieri, 1991 ).We evaluated the effect of freezing-thawing on tumor cells in PBSC grafts of breast cancer (BC) patients undergoing autologous PBSC transplantation. PBSC grafts obtained from 37 BC patients were tested for cytokeratin positive (CK+) cells by immunocytochemistry (ICC) staining using anti-cytokeratin antibodies ( A45B/B3, Epimet, Baxter) and APAAP technique both on cells spun directly onto a slide (cytospin) and on cells obtained after in vitro liquid culture (LC) assay. We have previously shown that LC assay in PBSC samples may enhance identification of CK+ cells surviving and growing under specific culture conditions, as compared to standard ICC staining on cytospin (Gibelli, 2000). When fresh cells were examined, we found CK+ cells in 9 cases out of 37 after LC assay, whereas only in 3 patients CK+ cells were found on cytospin ICC. Interestingly, after cryopreservation by freeze-controlled rate and rapid thawing, no CK+ cells were detected after LC assays in all 37 leukapheresis products. We then performed an experiment using a primary cell line of CK+ cells obtained from the pleural effusion of a BC patient. CK+ cells were added at 3 escalating concentrations ( l CK+ cell/ 50.000,100.000 and 200.000) to a PBSC sample obtained from a lymphoma patient (CK-). Before freezing, CK+ cell frequency ranged from 4/10' to 1.8/106 on cytospin ICC, and from 1/10" to 0.06/ 10 after LC assay (at higher and lower CK+ cell contamination, respectively). After the freezing/thawing procedure, the total cell viability was consistently 70%, and the recovery of CFU-GM and BFU-E was 70%, when compared to fresh PBSC. CK+ cells were still found in each thawed samples by ICC staining on cytospin (frequency ranging from 6/106 to 1.2/10 , respectively). Surprisingly, when testing thawed samples by LC assays, no viable CK+ cells were found in any sample. Taken these preliminary findings together with data obtained from PBSC grafts in 37 patients so far evaluated, we suggest that primary BC cells might be very sensitive to cryopreservation, and although present after thawing, they might loose their viability and their growth potential.
|Issue number||11 PART II|
|Publication status||Published - 2000|
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