Figure 4.
Correlation of HLA-DR expression with chromosome 6p CNA status in PCNSL. (A) Expression of HLA-DR in PCNSL was demonstrated by immunohistochemistry (IHC) in different genomic groups of 6p. Tumors with no loss at 6p or with hemizygous (1 copy) deletion at 6p21.3 exhibited stronger expression of HLA-DR than tumors with HD at 6p21.3 or CN-LOH at 6p. Original magnification, ×400. Scale bar, 20 μm. Hematoxylin and eosin (H&E) counterstain. (B) Expression of HLA-DR in PCNSL was demonstrated by in situ hybridization and colocalization with CD20 and CD68. Multiplex fluorescence in situ hybridization probes were used to colocalize coexpression of HLA-DR (red) with CD20 (green) in 30 diagnostic FFPE specimens. Original magnification, ×400. Scale bar, 20 μm. DAPI (4’,6-diamidino-2-phenylindole; blue) counterstain was used to localize nuclei. Additional example images are provided in supplemental Figure 9. (C) 6p CN-LOH with colocalization of HLA-DR (red) and the macrophage (MΦ) marker CD68 (green). CD68 IHC was used to localize tumor macrophages. (D) HLA-DR expression by mean fluorescence intensity (MFI) in different 6p CNA subgroups and infiltrating macrophages (CD68+) as a positive internal control. Negative HLA-DR coexpression by CD20+ lymphoma cells, defined as MFI 2 standard deviations below the mean MFI of HLA-DR expressed by tumor-associated macrophages (n = 5 cases), was detected in each of the 2 6p21.3 focal HD cases and in 9 of the 10 6p CN-LOH cases tested. Pairwise comparisons showed that the MFI of HLA-DR expression by lymphoma was significantly lower in PCNSL with 6p CN-LOH (n = 10) and 6p21.3 focal HD (n = 2) than with 6p hemizygous deletion (n = 9, P = .0007 and P = .00022, respectively), to 6p no loss (n = 9, P = .013 and P = .0029, respectively), and to CD68+ macrophages (P = .0022 and P = .0024, respectively). There was no significant difference in the MFI of HLA-DR expression by lymphoma cells among tumors with 6p hemizygous deletion, 6p no loss, and infiltrating macrophages, nor between 6p CN-LOH and 6p focal HD. P values are calculated from 2-sided t test. (E) Kaplan-Meier analysis demonstrates that PCNSL cases with negative tumor expression of HLA-DR exhibited shorter PFS than HLA-DR–positive PCNSL (P = .0058 by 2-sided log-rank test). Absent HLA-DR expression by lymphoma was associated with a trend toward shorter OS (data not shown).

Correlation of HLA-DR expression with chromosome 6p CNA status in PCNSL. (A) Expression of HLA-DR in PCNSL was demonstrated by immunohistochemistry (IHC) in different genomic groups of 6p. Tumors with no loss at 6p or with hemizygous (1 copy) deletion at 6p21.3 exhibited stronger expression of HLA-DR than tumors with HD at 6p21.3 or CN-LOH at 6p. Original magnification, ×400. Scale bar, 20 μm. Hematoxylin and eosin (H&E) counterstain. (B) Expression of HLA-DR in PCNSL was demonstrated by in situ hybridization and colocalization with CD20 and CD68. Multiplex fluorescence in situ hybridization probes were used to colocalize coexpression of HLA-DR (red) with CD20 (green) in 30 diagnostic FFPE specimens. Original magnification, ×400. Scale bar, 20 μm. DAPI (4’,6-diamidino-2-phenylindole; blue) counterstain was used to localize nuclei. Additional example images are provided in supplemental Figure 9. (C) 6p CN-LOH with colocalization of HLA-DR (red) and the macrophage (MΦ) marker CD68 (green). CD68 IHC was used to localize tumor macrophages. (D) HLA-DR expression by mean fluorescence intensity (MFI) in different 6p CNA subgroups and infiltrating macrophages (CD68+) as a positive internal control. Negative HLA-DR coexpression by CD20+ lymphoma cells, defined as MFI 2 standard deviations below the mean MFI of HLA-DR expressed by tumor-associated macrophages (n = 5 cases), was detected in each of the 2 6p21.3 focal HD cases and in 9 of the 10 6p CN-LOH cases tested. Pairwise comparisons showed that the MFI of HLA-DR expression by lymphoma was significantly lower in PCNSL with 6p CN-LOH (n = 10) and 6p21.3 focal HD (n = 2) than with 6p hemizygous deletion (n = 9, P = .0007 and P = .00022, respectively), to 6p no loss (n = 9, P = .013 and P = .0029, respectively), and to CD68+ macrophages (P = .0022 and P = .0024, respectively). There was no significant difference in the MFI of HLA-DR expression by lymphoma cells among tumors with 6p hemizygous deletion, 6p no loss, and infiltrating macrophages, nor between 6p CN-LOH and 6p focal HD. P values are calculated from 2-sided t test. (E) Kaplan-Meier analysis demonstrates that PCNSL cases with negative tumor expression of HLA-DR exhibited shorter PFS than HLA-DR–positive PCNSL (P = .0058 by 2-sided log-rank test). Absent HLA-DR expression by lymphoma was associated with a trend toward shorter OS (data not shown).

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