Molecular Signatures for Tumor Classification Yasin Mamatjan, Sameer Agnihotri, Anna Goldenberg, Peter Tonge, Sheila Mansouri, Gelareh Zadeh, Kenneth Aldape  The Journal of Molecular Diagnostics  Volume 19, Issue 6, Pages 881-891 (November 2017) DOI: 10.1016/j.jmoldx.2017.07.008 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 A: Similarity network fusion (SNF) cluster analysis showing the correlation of molecular platforms with tumor histology on the basis of 19 tissue types and their cluster membership for mRNA, methylation, miRNA, reverse-phase protein array (RPPA), and copy number variation (CNV). Not available data are white. Mixed clusters without a dominant tumor type (<50%) are black. B: The overlap among the subtypes based on mRNA expression, DNA methylation, and integrated mRNA and DNA methylation (iRM) obtained through SNF clustering (spectral clustering) of 19 cancer types and 5381 samples (based on the manual curation of iRM subtypes, which we called curated subtypes). The cluster sequence and sample memberships were sorted on the basis of the curated subtype. C: A comparison of subtype error rates for mRNA and methylation, showing the correlation of individual platform analysis with tumor subtype. D: Glioma subtype analysis based on mRNA, methylation, and iRM. The glioma subtypes were based on 322 samples with known IDH and 1p/19q status that resulted in 83%, 89%, and 99% concordance based on mRNA expression, DNA methylation, and iRM, respectively. These three subtypes are noted in sea green [IDH mutant, 1p/19q codeleted (IDHmut-codel) cluster], red [IDH wild-type (IDHwt) cluster], and blue [IDH mutant, 1p/19q non-codeleted (IDHmut-noncodel) cluster] for mRNA, methylation, and iRM, respectively. ACC, adrenocortical carcinoma; BLCA, urothelial bladder carcinoma; BRAF, B-Raf; BRCA, breast cancer; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; DLBC, diffuse large B-cell lymphoma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe renal cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; SKCM, skin cutaneous melanoma; THCA, papillary thyroid carcinoma. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A flow chart showing the process of identification and characterization of outliers based on similarity network fusion (SNF) clustering, random forest (RF) classification, and hierarchical clustering of mRNA and methylation data, in addition to further analysis of mutational data and miRNA based on SNF clusters. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: Hierarchical clustering of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and uterine carcinoma based on mRNA expression. B: Hierarchical clustering of CESC and uterine carcinoma based on DNA methylation. C: Hierarchical clustering of kidney renal papillary cell carcinoma (KIRP) and urothelial bladder carcinoma (BLCA) based on mRNA expression. D: Hierarchical clustering of KIRP and BLCA based on DNA methylation profile. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: Identification of a kidney renal papillary cell carcinoma (KIRP) outlier (ID: 7130) grouped with urothelial bladder carcinoma (BLCA). Mutational signatures of BLCA (red) and KIRP (sea green). The outlier has mutations in FGFR3, KDM6A, and STAG2. B: Histological examination of hematoxylin and eosin (H&E) sections for the outlier (ID: 7130), BLCA, and KIRP. C: Identification of a KIRP outlier (ID: 5892) grouped with kidney renal clear cell carcinoma (KIRC). Mutational signatures of KIRC (red), KIRP (sea green), and the outlier. BAP1 and PBRM1 gene mutations are mutually exclusive. The sample contained VHL and BAP1 mutations. D: Histological examination of H&E sections for the outlier (ID: 5892) and KIRC. E: Identification of a cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) outlier (ID: A3LA) grouped with the uterine carcinoma group. Mutational signatures of CESC (red), uterine carcinoma (sea green), and the outlier. The outlier contains mutations in TP53. F: Histological examination of H&E sections for the outlier (ID: A3LA), CESC, and uterine carcinoma. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 A generic procedure for tumor subtype identification. Single platform–based tumor type and subtype analysis using spectral clustering, followed by integrated multiplatform analysis [integrated mRNA and DNA methylation (iRM)]. We specifically investigated the association of subtypes of mRNA, methylation, and iRM with clinical data (survival) and exome mutation. CNV, copy number variation; RF, random forest; RPPA, reverse-phase protein array; SNF, similarity network fusion. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Pan-Cancer 12 analysis showing the correlation of molecular platforms with tumor histology on the basis of 12 tissue types and their cluster membership for cluster-of-clusters analysis (COCA), mRNA, methylation, miRNA, reverse-phase protein array (RPPA), and copy number variation (CNV). Not available data are white. Mixed clusters without a dominant tumor type (<50%) are black. BLCA, urothelial bladder carcinoma; BRCA, breast cancer; COAD, colon adenocarcinoma; GBM, glioblastoma; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; LAML, acute myeloid leukemia; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian carcinoma; READ, rectal adenocarcinoma; UCEC, uterine corpus endometrial carcinoma. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Two main subtypes of papillary thyroid carcinoma [THCA; namely, THCA–B-Raf (BRAF) and THCA–non-BRAF]. A: Mutation analysis for THCA subtypes based on mRNA. The percentages of mutations for THCA subtypes were calculated on the basis of 439 sequenced samples. We noted BRAF mutations in red and NRAS-HRAS-KRAS mutations in blue for mRNA, methylation, and integrated mRNA and DNA methylation (iRM). NRAS-HRAS-KRAS gene mutations are mutually exclusive in the THCA–non-BRAF subtype. B: Mutation analysis for THCA subtypes based on methylation. C: Mutation analysis for THCA subtypes based on iRM. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S4 A Kaplan-Meier curve showing the survival statistics of glioma subtypes. A: mRNA-based glioma subtypes. B: Methylation-based glioma subtypes. C: Integrated mRNA and DNA methylation (iRM)–based glioma subtypes. IDHmut-codel, IDH mutant, 1p/19q codeleted; IDHmut-noncodel, IDH mutant, 1p/19q non-codeleted; IDHwt, IDH wild-type. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S5 A: Mutational signatures of kidney renal clear cell carcinoma (KIRC) and kidney chromophobe renal cell carcinoma (KICH). B: Histological features of KIRC, KICH, and an outlier sample (ID: 3440). Original magnification: ×100 (B). H&E, hematoxylin and eosin. The Journal of Molecular Diagnostics 2017 19, 881-891DOI: (10.1016/j.jmoldx.2017.07.008) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions