Figure 1.
Figure 1. In situ analysis of normal and MF BM biopsy OCs. (A) Representative images of BM tissue from healthy controls (Ctrl; n = 3) and MF patients (n = 50) stained for TRAP and cathepsin K, with Hoechst 33258 as nuclear counterstain. As shown, few OCs are detected in normal BM biopsies, whereas abundant numbers of OCs are detected in the BM of MF patients. (B) Representative BM biopsy image (i) and OC quantitation (ii) in tissue sections from normal Ctrl and MF patients. Depicted BM section was stained for TRAP (green) and cathepsin K (red), with Hoechst 33258 as nuclear counterstain (blue). Whole-tissue image was assembled from 81 spectrally unmixed fields taken at a magnification of ×200, and a phenotype map of specimen was generated by a pattern-recognition algorithm. Red areas represent cellular BM, gray areas outline bone, and asterisks denote individual OCs. Bars represent median with 95% confidence interval (CI). (C) Comparison of OC numbers and BM fibrosis grade. As shown, MF patients with advanced-stage fibrosis (MF-3; n = 27) have higher numbers of OCs compared with patients with MF-1 (n = 4) and MF-2 (n = 13). BM fibrosis was independently graded using the European consensus criteria by pathologists who analyzed the patients’ BM biopsy specimens. Bars represent median with 95% CI. (D) A representative image of BM biopsy sections obtained from MF patients with a JAK2V617F mutation (n = 5). BM biopsy sections were immnuostained with antibodies against TRAP, cathepsin K, and total and phosphorylated (p) JAK2 protein. The cells’ nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images depict multiple OCs along thickened bone trabecula expressing high levels of pJAK2. At the time of biopsy, the patients’ peripheral blood JAK2V617F allele burden was 75.65%. (E) Analysis of randomly microdissected single OCs. (i) Representative image of BM biopsy sections obtained from CALR-mutated MF patients (n = 10) stained with anti-TRAP antibodies and hematoxylin QS nuclear counterstain. Dashed lines denote individual TRAP+ cells that were captured from the BM tissue sections by immunoguided laser microdissection. (ii) JAK2V617F (n = 6) and CALR exon 9 mutation (n = 10) allele burden in circulating low-density cells (LDC) and OCs randomly microdissected from the BM of mutation MF patients harboring 1 of those mutations. As shown, most single microdissected OCs expressed a higher mutant allele burden than LDC. Dashed lines denote samples with a decrease in allele burden. γ correction was not applied. Bars represent 100 μm (A,D,Ei) and 1 mm (Bi). ns, not significant.

In situ analysis of normal and MF BM biopsy OCs. (A) Representative images of BM tissue from healthy controls (Ctrl; n = 3) and MF patients (n = 50) stained for TRAP and cathepsin K, with Hoechst 33258 as nuclear counterstain. As shown, few OCs are detected in normal BM biopsies, whereas abundant numbers of OCs are detected in the BM of MF patients. (B) Representative BM biopsy image (i) and OC quantitation (ii) in tissue sections from normal Ctrl and MF patients. Depicted BM section was stained for TRAP (green) and cathepsin K (red), with Hoechst 33258 as nuclear counterstain (blue). Whole-tissue image was assembled from 81 spectrally unmixed fields taken at a magnification of ×200, and a phenotype map of specimen was generated by a pattern-recognition algorithm. Red areas represent cellular BM, gray areas outline bone, and asterisks denote individual OCs. Bars represent median with 95% confidence interval (CI). (C) Comparison of OC numbers and BM fibrosis grade. As shown, MF patients with advanced-stage fibrosis (MF-3; n = 27) have higher numbers of OCs compared with patients with MF-1 (n = 4) and MF-2 (n = 13). BM fibrosis was independently graded using the European consensus criteria by pathologists who analyzed the patients’ BM biopsy specimens. Bars represent median with 95% CI. (D) A representative image of BM biopsy sections obtained from MF patients with a JAK2V617F mutation (n = 5). BM biopsy sections were immnuostained with antibodies against TRAP, cathepsin K, and total and phosphorylated (p) JAK2 protein. The cells’ nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images depict multiple OCs along thickened bone trabecula expressing high levels of pJAK2. At the time of biopsy, the patients’ peripheral blood JAK2V617F allele burden was 75.65%. (E) Analysis of randomly microdissected single OCs. (i) Representative image of BM biopsy sections obtained from CALR-mutated MF patients (n = 10) stained with anti-TRAP antibodies and hematoxylin QS nuclear counterstain. Dashed lines denote individual TRAP+ cells that were captured from the BM tissue sections by immunoguided laser microdissection. (ii) JAK2V617F (n = 6) and CALR exon 9 mutation (n = 10) allele burden in circulating low-density cells (LDC) and OCs randomly microdissected from the BM of mutation MF patients harboring 1 of those mutations. As shown, most single microdissected OCs expressed a higher mutant allele burden than LDC. Dashed lines denote samples with a decrease in allele burden. γ correction was not applied. Bars represent 100 μm (A,D,Ei) and 1 mm (Bi). ns, not significant.

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