PubMed · 11652699
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Howard L Rosenberg. Informed consent.. https://pubmed.ncbi.nlm.nih.gov/11652699/
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BACKGROUND: Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. METHODS: We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients' primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. RESULTS: Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. CONCLUSIONS: Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.
A 5-year-old boy was admitted for anemia, thrombocytopenia, and azotemia. Bone marrow examination demonstrated only erythroid hyperplasia, and he was diagnosed with an atypical form of hemolytic uremic syndrome (HUS). He was treated with daily prednisolone, and hematological profiles and renal function normalized 1 month later. However, heavy proteinuria persisted, and renal biopsy findings were consistent with HUS. Four months later, pancytopenia developed with recurrence of azotemia, and a second bone marrow examination revealed acute lymphoblastic leukemia (ALL). The remission of proteinuria with no recurrences of HUS after antileukemic treatment suggests that the HUS was associated with the ALL.
Detection of genetic markers associated with early breast cancer may prove clinically relevant for identifying patients at increased risk for relapse. Loss of heterozygosity (LOH), a specific genetic aberration, commonly occurs during breast cancer initiation and metastasis. Early detection of tumor metastasis to bone marrow (BM) using conventional histochemical techniques has been limited because of suboptimal efficiency and sensitivity. Because bone is such a common site of breast cancer recurrence, we sought to determine whether microsatellite markers associated with breast cancer could be detected in BM aspirates from patients with early-stage breast cancer. Cell-free plasma from BM aspirates in 48 patients was assessed for LOH using a panel of eight polymorphic microsatellite markers. LOH was detected in 11 (23%) of 48 patients' BM aspirates. Advancing American Joint Committee on Cancer (AJCC) stage was associated with an increased incidence of LOH. Concordance was present between LOH identified in BM aspirates and matched-pair primary tumors. No samples contained detectable tumor cells on routine histology. In 24 patients, paired peripheral blood serum samples were available for analysis. In these cases, BM detection of LOH was more frequent than serum. This study demonstrates the novel finding of tumor-related genetic markers in BM aspirate plasma of early-stage breast cancer patients and provides a unique approach for assessing subclinical systemic disease progression and the monitoring of breast cancer patients.