title: Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT-PCR for cytokeratin-19 mRNA.
abstract: There is considerable interest in an autologous transplantation (AT) programme for patients with high-risk breast cancer; however, the issue of the incidence of occult bone marrow (BM) micrometastasis at diagnosis, and the cancer contamination of peripheral blood stem cell (PBSC) collections used for haematological rescue, is still debated. The presence of BM micrometastasis was evaluated in bilateral BM biopsies obtained at diagnosis of 33 patients with stage II/IIIA breast cancer using: (i) a 'nested' reverse transcriptase-polymerase chain reaction (RT-PCR) assay for cytokeratin 19 (K19) mRNA, (ii) histology, and (iii) immunohistochemistry (IHC) analysis with a panel of three monoclonal antibodies. The RT-PCR assay only was used to determine contamination of PBSC collections obtained after priming with recombinant human granulocyte-colony stimulating factor (rhG-CSF). K19 transcripts in one or both BM samples were detected in 48% of patients at diagnosis, with an overall 85% concordance with the results of IHC analysis. On the other hand, 56% of PCR- and IHC-positive BM samples were diagnosed as 'normal' on histological analysis. 57% of patients showed K19 mRNA in at least one PBSC collection; the possibility to have contaminated PBSC collections was significantly higher in patients with K19 positivity in BM at diagnosis. In four patients who had shown K19 positivity in BM and in PBSC collections, immunoselected CD34+ cells used for haematological rescue were K19-negative. There was a trend towards longer relapse free survival (RFS) in patients transplanted with K19-negative PBSC collections as compared to the others. In conclusion, a substantial proportion of patients with high-risk non-metastatic breast cancer present occult BM micrometastasis at diagnosis and also show cancer contamination of PBSC collections used for AT. These might represent a category of patients with poorer prognosis after AT, and possible candidates for more intensive and/or alternative therapeutic regimens, including AT with purged PBSCs.
authors.affiliation: Division of Haematology, University of Florence, Azienda Ospedaliera di Careggi, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Vannucchi
authors.forename: A
authors.initials: A
authors.lastname: Bosi
authors.forename: S
authors.initials: S
authors.lastname: Glinz
authors.forename: P
authors.initials: P
authors.lastname: Pacini
authors.forename: S
authors.initials: S
authors.lastname: Linari
authors.forename: R
authors.initials: R
authors.lastname: Saccardi
authors.forename: R
authors.initials: R
authors.lastname: Alterini
authors.forename: L
authors.initials: L
authors.lastname: Rigacci
authors.forename: S
authors.initials: S
authors.lastname: Guidi
authors.forename: L
authors.initials: L
authors.lastname: Lombardini
authors.forename: G
authors.initials: G
authors.lastname: Longo
authors.forename: M P
authors.initials: MP
authors.lastname: Mariani
authors.forename: P
authors.initials: P
authors.lastname: Rossi-Ferrini
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis; D019046:Bone Marrow Neoplasms / Q000737:chemistry / Q000556:secondary*; D001943:Breast Neoplasms / Q000473:pathology*; D018572:Disease-Free Survival; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor / Q000627:therapeutic use; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007633:Keratins / Q000032:analysis*; D007937:Leukapheresis; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity; D016896:Treatment Outcome
chemical_list: D018952:Antigens, CD34; D012333:RNA, Messenger; D016179:Granulocyte Colony-Stimulating Factor; D007633:Keratins