Five micrometer paraformaldehyde-fixed, paraffin-embedded tumor sections were stained with hematoxylin and eosin and histologically classified. A. Papillary carcinoma B. Squamous cell carcinoma C. Pituitary adenoma D. Malignant peripheral nerve sheath tumor. Bars = 50 mm.

A. Schematic of mouse chromosome 11 depicting the positions of the microsatellite markers and SNP assessed relative to Trp53 and Nf1. Microsatellite markers D11Mit29, D11Mit31, and D11Mit219 were used to assess Trp53 and the intervening region between Trp53 and Nf1 in radiation-induced tumors and normal tail controls. B. Fragment analysis of the D11Mit29 locus reveals that normal tissue from C57Bl/6 (B6) and 129/Sv (129) mice show discrete peaks at 141 and 147/149, respectively, and these allelic contributions are readily visualized in F1 mice. In radiation-induced osteosarcoma tumor (OS, an osteosarcoma arising in a Nf1 mutant mouse receiving 30 Gy CI) DNA the B6 fragment is significantly reduced (*) compared to control tail DNA. C. SNP rs13481119 was sequenced, showing heterozygosity in F1 mice. Comparison of tumor DNA to matched tail shows intact heterozygosity in SCCA (a squamous cell carcinoma), reduction of B6 peak in OS, and reduction of 129 peak in a pituitary adenoma. D. The pattern of LOH is described for each microsatellite and SNP locus by tumor type arising in Nf1 mutant mice. The number of tumors losing the C57Bl/6 allele (table on left) or 129/Sv allele (table on right) is listed over the total number of tumors assessed at the locus.

Kaplan-Meier survival curves were used to depict overall survival, solid tumor free survival, and hematologic disease free survival in WT and Nf1 mutant mice. Log-rank tests were used to test for differences between survival curves. The latency to death was measured from the date of the last radiation fraction. A and B. WT and Nf1 mutant mice had decreased survival after CI. C and D. Kaplan-Meier cause-specific survival curves were generated to reflect death due to hematologic malignancies or solid tumors. Nf1^+/− mice exposed to 15 Gy CI were significantly more likely to die with hematologic disease than those that were unirradiated or exposed to 30 Gy CI (HR 4.151, 95% CI: 0.9081–18.971, p=0.0465). D. Nf1^+/− mice exposed to 30 Gy CI were significantly more likely to die of solid tumors than mice that were unirradiated or exposed to 15 Gy CI (HR 1.659, 95% CI: 0.3037 – 9.058, p=0.04).

Five micrometer paraformaldehyde-fixed, paraffin-embedded sections of spleen and sternum were stained with hematoxylin and eosin. A. Box plots of hemoglobin levels at the time of sacrifice for each treatment group. Nf1 mutant mice exposed to 15 Gy CI are significantly more anemic compared to Nf1 mutant mice that are unirradiated or exposed to 30 Gy CI, and compared to all WT mice (p<0.05, two-tailed test). Thick black line indicates median, upper edge of box indicates third quartile, lower edge of box indicates first quartile. B. Box plots of hemoglobin levels were compared based on cause of death. Mice sacrificed due to hematologic disease were anemic compared to those who did not have histologic evidence of hematologic disease (p<0.001, two tailed test), while no significant difference in hemoglobin levels was observed between mice that did and did not die of solid tumor. C. Top row, left to right: Normal spleen (20X and 100X), and bone marrow (100X) from an unirradiated Nf1 mutant mouse. Middle row, left to right: Spleen (20X and 100X) and bone marrow (100X) from an irradiated Nf1 mutant mouse with peripheral anemia. Bottom row, left to right: Spleen (20X and 100X) and bone marrow (100X) from an irradiated Nf1 mutant mouse that developed anemia but whose spleen and marrow lacked erythropoietic expansion.

Five micrometer tumor sections were stained with DAPI and phospho-specific antibodies were used to visualize in vivo levels of pS6 and pAkt. A. Tumor sections were visualized with fluorescence microscopy and six 40X fields from each section were scored in blinded fashion as either 1 (no signal), 2 (low signal) or 3 (high signal), then averaged. Average scores for pS6 and pAkt are plotted. Tumors in which LOH at SNP rs13481119 (intragenic to Nf1) were assessed are indicated as follows: tumors showing intact heterozygosity at SNP rs13481119 are plotted as light blue circles, with LOH of B6 allele as dark blue squares, or with LOH of 129 allele as magenta squares. B. Phosphorylated S6 (pS6) levels are elevated in tumor sections from a radiation-induced pituitary adenoma and osteosarcoma, while pAkt levels are not correspondingly elevated. Bar = 50 μm.