Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1337-4) contains supplementary material, which is available to authorized users

Enhancement of the signal-to-noise ratio in (H2O)-O-15 bolus PET activation images: A combined cold-bolus, switched protocol

To increase the signal-to-noise ratio (S/N) of (H2O)-O-15 bolus PET activation images, we designed and tested a data acquisition protocol that alters the relative distribution of tracer in the uptake and washout phases of the input function. This protocol enhances the S/N gains obtained with conventional switched protocols by combining task switching and the use of a large bolus of blood free of tracer (cold bolus). The cold bolus is formed by sequestering blood in the lower limbs with a double cuff before tracer injection. Methods: The effect of a combined cold-bolus, switched protocol on the signal from activation images was first simulated using a compartmental model of the uptake of (H2O)-O-15 into the brain. Then, the effectiveness of the protocol was investigated in 4 healthy volunteers performing a language task. Each volunteer underwent scanning 12 times: 3 activation/baseline and 3 baseline/activation scans using the conventional switched protocol and 3 activation/baseline and 3 baseline/activation scans using the combined cold-bolus, switched protocol. The S/N changes introduced when using the cold bolus were analyzed by comparing, across protocols, the magnitude and statistical significance of the activation foci associated with the execution of the language task identified in the averaged subtracted images, and by comparing image noise levels. Results: In the simulated datasets, the combined protocol yielded a substantial increase in the activation signals for scan durations greater than 60 s, in comparison with equivalent signals yielded by the switched protocol alone. In the PET experiments, activation foci obtained using the combined protocol had significantly higher t statistic values than did equivalent foci detected using the conventional switched protocol (mean improvement, 36%). Analysis of the S/N in the averaged subtracted images revealed that the improvements in statistical significance of the activation foci were caused by increases in the signal magnitudes and not by decreases in overall image noise. Conclusion: We designed a data acquisition protocol for (H2O)-O-15 bolus PET activation studies that combines the use of a tracer-free bolus with a switched protocol. Simulated and experimental data suggest that this combined protocol enhances the S/N gains obtained with a conventional switched protocol. Implementation of the combined protocol in (H2O)-O-15 bolus activation studies was easy

Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma.

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease