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PubMed · 7136467

Human bone marrow sinus: a scanning electron microscopic study.

Y Hirasawa·H Tokuhiro·F Kuto·T Nagaoka·M Hayashi·E Miwa
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Bone MarrowBone Marrow CellsCell MovementHematopoiesisHumansMicroscopy, Electron, ScanningJournal Article

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Y Hirasawa, H Tokuhiro, F Kuto, T Nagaoka, M Hayashi, E Miwa. 1982. Human bone marrow sinus: a scanning electron microscopic study.. https://pubmed.ncbi.nlm.nih.gov/7136467/

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Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow.

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.

Bone Marrow↗
PubMed1996

P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells.

P-selectin glycoprotein ligand-1 (PSGL-1) is a muclin-like glycoportein ligand for P- and E-selectin on myeloid cells and a subset of lymphocytes. We used flow cytometry and immunohistochemistry to examine expression of PSGL-1 on minor leukocyte populations, differentiating hematopoletic cells, and nonhematopoietic tissues using two monoclonal antibodies to distinct protein epitopes on PSGL-1. In the bone marrow, PSGL-1 was expressed on myeloid cells from the myeloblast stage to the segmented neutrophil, but was not detected on erythroblasts or megakaryocytes. All types of circulating myeloid cells expressed PSGL-1, and PSGL-1 was retained after extravasation of myetoid cells into tissues. PSGL-1 was also expressed on circulating dendritic cells, monocyte-derived dendritic cells, dendritic cells in lymphoid tissues and epidermis, and follicular dendritic cells. All types of lymphoid cells examined expressed PSGK-1, including immature and mature thymocytes, naive and memory T cells, gamma/delta T cells, netural killer cells, B cells and CD34+ progenitor cells. However, PSGL-1 levels were substantially lower on tonsillar lymphocytes than on circulating lymphocytes, suggesting that PSGL-1 expression is down regulated during or after entry of lymphocytes into secondary lymphoid tissue. Although PSGL-1 antigen was detected primarily on hamatopoietic cells, it was also present on time epithelium of the fallopian tube. Furthermore, PSGL-1 antigen gen was detected sporadically on microvascular endothelium in some pathologic tissues. This suggests that PSGL-1 may have functions other than mediating leukocyte adhersion.

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PubMed1996

Minimal disease detection in patients with alveolar rhabdomyosarcoma using a reverse transcriptase-polymerase chain reaction method.

BACKGROUND: Polymerase chain reaction (PCR) assays that detect fusion genes resulting from consistent chromosomal translocations have been used to detect minimal residual disease, primarily in hematologic malignancies. Molecular assays have been developed recently that detect the PAX3-FKHR or PAX7-FKHR fusion transcript resulting from the t(2; 13) or t(1; 13) translocation consistently observed in alveolar rhabdomyosarcoma. Because of the tumor's propensity to disseminate widely, our aim was to determine whether or not reverse transcriptase-PCR assays could detect submicroscopic disease in bone marrow or peripheral blood specimens. METHODS: We analyzed 19 bone marrow samples from 11 patients with a known gene fusion in their primary tumor. Specimens were collected at diagnosis, remission, and relapse. Fourteen peripheral blood samples were obtained serially from 5 of these patients. A reverse transcriptase-PCR assay was used to detect the presence of the PAX3-FKHR or PAX7-FKHR fusion transcript. These results were compared with the results of microscopic examination of the bone marrow. Medical records of all 11 patients were reviewed. RESULTS: Adequate amplifiable RNA was obtained in 17 of 19 marrow samples. The PCR assay detected fusion products in all 4 specimens obtained from 3 patients with histologic evidence of bone marrow involvement. In addition, PAX3-FKHR fusion products were detected in bone marrows from 2 patients for whom there was no histologic evidence of disease. The fusion transcripts were not detected in any of the peripheral blood samples. CONCLUSIONS: The detection of submicroscopic disease is possible in alveolar rhabdomyosarcoma patients using PCR. These assays may have a role in staging, monitoring therapy, purging protocols for autologous bone marrow transplantation, and post therapy follow-up. Larger prospective studies are needed to address the clinical relevance of these results.

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