Revert oncoprint header removal, fix typo N=, update tumor purity sen…

…tence

content/03.results.md

@@ -117,20 +117,29 @@ Unless otherwise noted, each analysis was performed for diagnostic tumors using
 SNV consensus calling (**Figure {@fig:S1}** and **Figure {@fig:S2}A-G**) revealed, as expected, lower tumor mutation burden (TMB) (**Figure {@fig:S2}H**) in pediatric tumors compared to adult brain tumors from The Cancer Genome Atlas (TCGA) (**Figure {@fig:S2}I**), with hypermutant (> 10 Mut/Mb) and ultra-hypermutant (> 100 Mut/Mb) tumors [@doi:10.1016/j.cell.2017.09.048] only found within HGGs and embryonal tumors.
 **Figure {@fig:Fig2}** and **Figure {@fig:S3}B** depict oncoprints recapitulating known histology-specific driver genes in primary tumors across OpenPBTA histologies, and **Table S2** summarizes all detected alterations across cancer groups.
 
+#### Low-grade gliomas
+
 As expected, most (62%, 140/226) LGGs harbored a somatic alteration in _BRAF_, with canonical _BRAF::KIAA1549_ fusions as the major oncogenic driver [@doi:10.1186/s40478-020-00902-z] (**Figure {@fig:Fig2}A**). <!--SAMPLECOUNT-->
 We observed additional mutations in _FGFR1_ (2%), _PIK3CA_ (2%), _KRAS_ (2%), _TP53_ (1%), and _ATRX_ (1%) and fusions in _NTRK2_ (2%), _RAF1_ (2%), _MYB_ (1%), _QKI_ (1%), _ROS1_ (1%), and _FGFR2_ (1%), concordant with previous studies reporting near-universal upregulation of the RAS/MAPK pathway in LGGs [@doi:10.1186/s40478-020-00902-z; @doi:10.1016/j.ccell.2020.03.011].
 Indeed, gene set variant analysis (GSVA) revealed significant upregulation (ANOVA Bonferroni-corrected p < 0.01) of the KRAS signaling pathway in LGGs (**Figure {@fig:Fig5}B**).
 
+#### Embryonal tumors
+
  <!--SAMPLECOUNT-->
 Most (N = 95) embryonal tumors were medulloblastomas from four characterized molecular subtypes (WNT, SHH, Group3, and Group 4; see **Molecular Subtyping of CNS Tumors**), as identified by subtype-specific canonical mutations (**Figure {@fig:Fig2}B**).
 We detected canonical _SMARCB1/SMARCA4_ deletions or inactivating mutations in atypical teratoid rhabdoid tumors (ATRTs; **Table S2**) and C19MC amplification in ETMRs (displayed within "Other embryonal tumors" in **Figure {@fig:Fig2}B**) [@doi:10.1007/s00401-020-02182-2; @doi:10.1093/neuonc/noab178; @doi:10.1186/s40478-020-00984-9; @doi:10.1038/nature22973].
 
+#### High-grade gliomas
+
+
 <!--SAMPLECOUNT-->
-Across HGGs, _TP53_ (57%, 36/63) and _H3F3A_ (54%, 34/63) were both most mutated and co-occurring genes (**Figure {@fig:Fig2}A and C**), followed by frequent mutations in _ATRX_ (29%, 18/63) which is  commonly mutated in gliomas [@doi:10.1080/14728222.2018.1487953].
+Across HGGs, _TP53_ (57%, 36/63) and _H3F3A_ (54%, 34/63) were both most mutated and co-occurring genes (**Figure {@fig:Fig2}A and C**), followed by frequent mutations in _ATRX_ (29%, 18/63) which is commonly mutated in gliomas [@doi:10.1080/14728222.2018.1487953].
 We observed recurrent amplifications and fusions in _EGFR_, _MET_, _PDGFRA_, and _KIT_, highlighting that these tumors leverage multiple oncogenic mechanisms to activate tyrosine kinases, as previously reported [@doi:10.1002/ijc.32258; @doi:10.1016/j.ccell.2017.08.017; @doi:10.1186/s40478-020-00905-w].
 GSVA showed upregulation (ANOVA Bonferroni-corrected p < 0.01) of DNA repair, G2M checkpoint, and MYC pathways as well as downregulation of the TP53 pathway (**Figure {@fig:Fig5}B**).
 The two ultra-hypermutated tumors (> 100 Mutations/Mb) were from patients with mismatch repair deficiency syndrome [@doi:10.1093/neuonc/noz192].
 
+#### Other CNS tumors
+
 <!--SAMPLECOUNT-->
 We observed that 25% (15/60) of ependymomas were _C11orf95::RELA_ (now, _ZFTA::RELA_) fusion-positive [@doi:10.1038/nature13109] and 68% (21/31) of craniopharyngiomas contained _CTNNB1_ mutations (**Figure {@fig:Fig2}D**).
 We observed somatic mutations or fusions in _NF2_ in 41% (7/17) of meningiomas, 5% (3/60) of ependymomas, and 25% (3/12) of schwannomas, as well as rare fusions in _ERBB4_, _YAP1_, and/or _QKI_ in 10% (6/60) of ependymomas.
@@ -142,7 +151,7 @@ DNETs harbored alterations in MAPK/PI3K pathway genes, as was previously reporte
 
 ### Mutational co-occurrence, CNV, and signatures highlight key oncogenic drivers
 
-We analyzed mutational co-occurrence across the OpenPBTA, using a single tumor from each patient (N = 688) with WGS. <!--SAMPLECOUNT-->
+We analyzed mutational co-occurrence across the OpenPBTA, using a single tumor from each patient (N = 668) with WGS. <!--SAMPLECOUNT-->
 The top 50 mutated genes (see **STAR Methods** for details) in primary tumors are shown in **Figure {@fig:Fig3}** by tumor type (**A**, bar plots), with co-occurrence scores illustrated in the heatmap (**B**).
 As expected, _TP53_ was the most frequently mutated gene across the OpenPBTA (8.7%, 58/668), significantly co-occurring with _H3F3A_ (OR = 30.05, 95% CI: 14.5 - 62.3, q = 2.34e-16), _ATRX_ (OR = 23.3, 95% CI: 9.6 - 56.3, q = 8.72e-9), _NF1_ (OR = 8.26, 95% CI: 3.5 - 19.4, q = 7.40e-5), and _EGFR_ (OR = 17.5, 95% CI: 4.8 - 63.9, q = 2e-4), with all of these driven by HGGs and consistent with previous reports [@doi:10.1016/j.ccell.2017.08.017; @doi:10.1093/neuonc/noaa251; @doi:10.1038/ng.2938].
 
@@ -276,7 +285,7 @@ We additionally explored the ratio of CD8+ to CD4+ T cells across tumor subtypes
 This ratio has been associated with better immunotherapy response and prognosis following PD-L1 inhibition in non-small cell lung cancer or adoptive T cell therapy in multiple stage III or IV cancers [@doi:10.1136/jitc-2021-004012; @doi:10.4236/jct.2013.48164].
 While adamantinomatous craniopharyngiomas and Group 3 and Group 4 medulloblastomas had the highest ratios (**Figure {@fig:S6}F**), very few tumors had ratios greater than 1, highlighting an urgent need to identify novel therapeutics for pediatric brain tumors with poor prognosis.
 
-Finally, we explored the potential influence of tumor purity by repeating selected transcriptomic analyses restricted to samples with high tumor purities within their cancer group(see **STAR Methods**).
-These new analyses were broadly consistent (**Figure {@fig:S7}D-I**) with results derived from all stranded RNA-Seq samples.
+Finally, we explored the potential influence of tumor purity by repeating selected transcriptomic analyses restricted to only samples with high tumor purity (see **STAR Methods**).
+Results from these analyses were broadly consistent (**Figure {@fig:S7}D-I**) with results derived from all stranded RNA-Seq samples.
 
 ![**Transcriptomic and immune landscape of pediatric brain tumors** A, First two dimensions from UMAP of transcriptome data. Points colored by broad histology. B, Heatmap of with significant GSVA scores for Hallmark gene sets with tumors ordered by cancer group. C, Box plots of quanTIseq estimates of immune cell proportions in select cancer groups with N > 15 tumors. Note: other HGGs and other LGGs have immune cell proportions similar to DMG and pilocytic astrocytoma, respectively, and are not shown. D, Forest plot depicting additive effects of _CD274_ expression, immune cell proportion, and extent of tumor resection on OS of medulloblastoma patients. HRs with 95% confidence intervals and p-values (multivariate Cox) are listed. Significant p-values are denoted with black diamonds. Reference groups are denoted by grey diamonds. Note: the Macrophage M1 HR was 0 (coefficient = -9.90e+4) with infinite upper and lower CIs, and thus was not included in the figure. E, Box plot of _CD274_ expression (log<sub>2</sub> FPKM) for medulloblastomas grouped by subtype. Bonferroni-corrected p-values from Wilcoxon tests are shown. Box plot represents 5% (lower whisker), 25% (lower box), 50% (median), 75% (upper box), and 95% (upper whisker) quantiles. Only stranded RNA-Seq data is plotted.](https://raw.githubusercontent.com/AlexsLemonade/OpenPBTA-analysis/37ec62fdc2fd9ff157f2f2c10b69e9bb36673363/figures/pngs/figure5.png?sanitize=true){#fig:Fig5 width="7in"}