Decreased expression of Numb, resulting in activation of the proto

SUMMARY:A human tumor xenograft (L56Br-X1) was established from a breast cancer axillary lymph node metastasis of a 53-year-old woman with a BRCA1 germ-line nonsense mutation (1806CϾT; Q563X), and a cell line (L56Br-C1) was subsequently derived from the xenograft. The xenograft carries only the mutant BRCA1 allele and expresses mutant BRCA1 mRNA but no BRCA1 protein as determined by immunoprecipitation or Western blotting. The primary tumor, lymph node metastasis, and xenograft were hypodiploid by DNA flow cytometry, whereas the cell line displayed an aneuploidy apparently developed via polyploidization. Cytogenetic analysis, spectral karyotyping, and comparative genomic hybridization of the cell line revealed a highly complex karyotype with numerous unbalanced translocations. The xenograft and cell line had retained a somatic TP53 missense mutation (S215I) originating from the primary tumors, as well as a lack of immunohistochemically detectable expression of steroid hormone receptors, epidermal growth factor receptor, human epidermal growth factor receptor 2 (HER-2), and keratin 8. Global gene expression analysis by cDNA microarrays supported a correlation between the expression profiles of the primary tumor, lymph node metastasis, xenograft, and cell line. We conclude that L56Br-X1 and L56Br-C1 are useful model systems for studies of the pathogenesis and new therapeutic modalities of BRCA1-induced human breast cancer. (Lab Invest 2003, 83:387-396).

BackgroundDisseminated tumour cells (DTCs) in the bone marrow of patients with breast cancer have been identified as an independent predictor of poor prognosis in patients with non-metastatic disease. This prospective study aimed to evaluate the presence and prognostic value of DTCs in the bone marrow of female patients with primary breast cancer.MethodsBetween 1999 and 2003, bone marrow aspirates were obtained from patients at the time of surgery for primary invasive breast cancer. DTCs in bone marrow were identified using monoclonal antibodies against cytokeratins for detection of epithelial cells. The detection of DTCs was related to clinical follow-up with distant disease-free survival (DDFS) and breast cancer-specific survival as endpoints. Bone marrow aspirates from adult healthy bone marrow donors were analysed separately.ResultsDTCs were analysed in 401 patients, and cytokeratin-positive cells were found in 152 of these (38%). An immunofluorescence (IF) staining procedure was used in 327 patients, and immunocytochemistry (IC) was performed in 74 patients. The IF-based method resulted in 40% DTC-positive cases, whereas 30% were positive using IC (p = 0.11). The presence of DTCs in bone marrow was not significantly related to patient or tumour characteristics. The presence of DTCs was not a prognostic factor for DDFS (IF: hazards ratio [HR], 2.2; 95% confidence interval [CI], 0.63–2.2; p = 0.60; IC: HR, 0.84; 95% CI, 0.09–8.1; p = 0.88). Significant prognostic factors were lymph node metastases, oestrogen receptor positivity, Nottingham histological grade, and tumour size using Cox univariate analysis. The analyses were positive for epithelial cells in bone marrow from adult healthy donors in 19 (25%) samples.ConclusionsThe detection of DTCs in bone marrow in primary breast cancer was previously shown to be a predictor of poor prognosis. We were not able to confirm these results in a prospective cohort including unselected patients before the standard procedure was established. Future studies with a standardised patient protocol and improved technique for isolating and detecting DTCs may reveal the clinical applications of DTC detection in patients with micrometastases in the bone marrow.

Multiple biopsies of normal breast tissue from 10 BRCA1 mutation carriers have been analyzed using array-based comparative genomic hybridization. Normal breast tissue from five age-matched control subjects without a family history of breast cancer was included for reference purposes. We repeatedly found multiple low copy number aberrations at a significantly higher frequency in histopathologically normal tissue from BRCA1 mutation carriers than in normal control tissue. Some of these aberrations were similar across samples from different patients and linked to biological functions such as transcriptional regulation and DNA binding. We also observed a high degree of genomic heterogeneity between samples from the same patient, suggestive of tissue heterogeneity and etiological clonality in the breast epithelium. We show that neither loss of heterozygosity nor promoter methylation of the wild-type BRCA1 allele is the predominant mechanistic origin of the observed genomic instability. Instead, we propose that haploinsufficiency of BRCA1 might be the underlying cause responsible for initiation of breast cancer in these predisposed women, making cells vulnerable to mitotic recombination. We also propose that loss of ERalpha expression is preceded by genetic instability in the initiation of BRCA1-dependent tumorigenesis, indicating that the breast epithelium of BRCA1 mutation carriers may initially be estrogen-responsive. Our results imply that genomic instability instigated by BRCA1 haploinsufficiency may be required for breast cancer initiation in BRCA1 mutation carriers. Finding molecular markers of tumor initiation and progression, for the potential use in early disease detection, may be of great clinical importance for the improved management of at-risk women.

Resistance to the HER-2 targeting drug trastuzumab is observed clinically, but the lack of suitable experimental models hampers studies of resistance mechanisms. We characterized a HER-2 positive carcinoma cell line (JIMT-1) derived from a 62-year-old breast cancer patient, clinically resistant to trastuzumab. m-FISH revealed a complex hyperdiploid karyotype with numerous marker chromosomes and unbalanced translocations. CGH revealed numerous regions of copy number aberration (CNA). Further analysis by a-CGH identified 27 regions of CNA (16 amplified, 11 deleted). 38% of the genes in the amplified regions were overexpressed, compared to only 9% in regions of normal copy number ratios (CNR). Accordingly, 26% of the genes in the deleted regions were underexpressed, compared to 10% in regions of normal CNR. Most amplified and overexpressed genes were located on chromosome 1 as well as on 8q, 12q14.1, 17q11-q21, and 20q13. In 17q11-q21 we identified two separate amplicons, the HER-2 amplicon, and a previously unreported amplicon at 17q21.31. Several aberrant genes are implicated in cancer development (e.g. JUN, CDK4 and SLUG protooncogenes, and the drug/hormone metabolizing genes GSTM1 and CYP24). We conclude that cytogenetic and expression profiling of JIMT-1 revealed several new features that needs further characterization, which may shed light on the trastuzumab resistance.4