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Table S4

AACR GENIE Data Guide

Supplemental Methods. Supplemental Table 1: ââ,¬â€¹Genomic Data Characterization by Center. Supplemental Table 2: ââ,¬â€¹Gene Panels Submitted by Each Center. Figure S1: Number of putative germline SNPs per sample, before and after uniform germline filtering. Figure S2ââ,¬â€¹. Distribution of total somatic mutation burden per sample stratified by sequencing panel. Figure S3: ââ,¬â€¹Log-scale comparison of mutation frequencies at hotspot sites between GENIE (data aggregated from all sequencing panels) and cancerhotspots.org (CHS) using a binomial test. Figure S4:ââ,¬â€¹ Comparison of mutation frequencies at hotspot sites in each GENIE sequencing panel with cancerhotspots.org (CHS) using a binomial test.

Supplementary Figure S3. Pathway annotation for HNSCC super-groups. Each horizontal bar graph represents enriched pathways in different subtypes. The number at the end of bar is the total number of genes in a pathway. Percentage of genes in the dataset that overlap with the pathway are shown in grey colour of the bar. The trend line represents the -log(p-value).

E2F4 gene signature modulation and association with overall survival in ER+/HER2- breast cancer treated with endocrine therapy. a) E2F4 gene signature expression at baseline (pre) and after 2-weeks of aromatase inhibitor treatment (post), data from ACOSOGZ1031b study. Tumors were divided according to the E2F4 score at baseline in high, medium or low tertiles. After two-weeks of aromatase inhibitor treatment, there was a decrease in the E2F4 score in all groups. However, the proportion of tumors still expressing a high post-treatment E2F4 score was greater in patients with a high E2F4 score at baseline (60%) than the other groups. b) Overall survival in patients with ER+ breast cancer treated with adjuvant endocrine therapy in the METABRIC database (n=1498) according to E2F4 signature score tertiles. c) Overall survival in patients with ER+ tumors from METABRIC according to E2F4 score tertiles in Luminal A and d) Luminal B tumors.

Purpose: Current classification of head and neck squamous cell carcinomas (HNSCC) based on anatomic site and stage fails to capture biologic heterogeneity or adequately inform treatment.Experimental Design: Here, we use gene expression-based consensus clustering, copy number profiling, and human papillomavirus (HPV) status on a clinically homogenous cohort of 134 locoregionally advanced HNSCCs with 44% HPV+ tumors together with additional cohorts, which in total comprise 938 tumors, to identify HNSCC subtypes and discover several subtype-specific, translationally relevant characteristics.Results: We identified five subtypes of HNSCC, including two biologically distinct HPV subtypes. One HPV+ and one HPV− subtype show a prominent immune and mesenchymal phenotype. Prominent tumor infiltration with CD8+ lymphocytes characterizes this inflamed/mesenchymal subtype, independent of HPV status. Compared with other subtypes, the two HPV subtypes show low expression and no copy number events for EGFR/HER ligands. In contrast, the basal subtype is uniquely characterized by a prominent EGFR/HER signaling phenotype, negative HPV-status, as well as strong hypoxic differentiation not seen in other subtypes.Conclusion: Our five-subtype classification provides a comprehensive overview of HPV+ as well as HPV− HNSCC biology with significant translational implications for biomarker development and personalized care for patients with HNSCC. Clin Cancer Res; 21(4); 870–81. ©2014 AACR.

Supplemental Table S1: The Preoperative Endocrine Prognostic Index (PEPI score) Supplemental Table S2:* Baseline Ki67: 7 patients had missing values Supplemental Table S3a: Driver Mutations Supplemental Table S3b: Driver Copy Number Alterations Supplemental Table S3c: Mutations clinically actionable. Drug prescription Supplemental Table S4: Actionable CNAs. Drug prescription Supplemental Table S5: Overlap between genes upregulated in resistant tumors and transcription factor targets genes Suplemental Table S6: Change from baseline to surgery in gene expressions of the 47 most upregulated genes in the tumors resistant to prolonged neoadjuvant letrozole from 60 HR-positive/HER2-negative breast cancers in POP trial. A decreased gene expression translates into a geometric mean change < 1 . An increased or stable gene expression translates into a geometric mean change {greater than or equal to} 1. Supplemental Table S7: Mutations detected in this study(post-filtering) Supplemental Table S8: Gene expression values (FPKM)

Supplementary data information

Supplementary Table S2. Centroids of the 821 gene signature for the three super-groups

PIK3CA mutations are associated with resistance to HER2-targeted therapies. We previously showed that HER2+/PIK3CAH1047R transgenic mammary tumors are resistant to the HER2 antibodies trastuzumab and pertuzumab but respond to PI3K inhibitor buparlisib (TPB). In this study, we identified mechanisms of resistance to combined inhibition of HER2 and PI3K. TPB-resistant tumors were generated by treating HER2+/PIK3CAH1047R tumor-bearing mice long term with the drug combination. RNA sequencing of TPB-resistant tumors revealed that extracellular matrix and cell adhesion genes, including collagen II (Col2a1), were markedly upregulated, accompanied by activation of integrin β1/Src. Cells derived from drug-resistant tumors were sensitive to TBP when grown in vitro, but exhibited resistance when plated on collagen or when reintroduced into mice. Drug resistance was partially reversed by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoate. Inhibition of integrin β1/Src blocked collagen-induced resistance to TPB and inhibited growth of drug-resistant tumors. High collagen II expression was associated with significantly lower clinical response to neoadjuvant anti-HER2 therapy in HER2+ breast cancer patients. Overall, these data suggest that upregulation of collagen/integrin/Src signaling contributes to resistance to combinatorial HER2 and PI3K inhibition. Cancer Res; 77(12); 3280–92. ©2017 AACR.

Supplementary Figure 1. HER2+/PIK3CAH1047R mammary tumors acquire resistance to trastuzumab + pertuzumab + BKM120 in vivo. Supplementary Figure 2. Primary tumor cells (PTCs) derived from TPB-resistant tumors are sensitive to TPB in vitro. Supplementary Figure 3. Collagen reduces sensitivity to TPB in human breast cancer cells. Supplementary Figure 4. TbRI inhibition does not affect Collagen-induced TPB resistance. Supplementary Figure 5. Collagen deposition is enhanced in residual drug-resistant tumors following neoadjuvant anti-HER2 therapy.

Supplementary Table 1. List of antibodies used for immunoblotting. Supplementary Table 2. Patient biopsies used for IHC analysis. Supplementary Table 3. List of genes that are significantly upregulated in TPB-resistant tumors relative to untreated parental tumors. Supplementary Table 4. Expression of ECM/cell adhesion genes in pre-treatment tumor biopsies from patients in the NeoSphere trial.

Supplemental Materials and Legend. Supplemental Experimental Procedures: Determination of HPV status; Gene expression profiling; Copy number aberration (CNA) experiment; Copy number aberration (CNA) analysis; Published Data Set Collection and Integration; Unsupervised Discovery of Gene Expression-Based Subtypes and Derivation of Gene Signatures; Validation of HNSCC Classification; Single Sample Gene Set Enrichment Analysis (ssGSEA); Pathway Analysis; Survival Analysis; Immunohistochemistry and Immunofluorescence. Supplementary Figure Legends. Supplementary Table S1. Data set source and group allocation Supplementary Table S3. Significant copy number gain and loss in the discovery set (101 samples and 75 genes). Supplementary Table S4. Significant copy number gain and loss in the validation set (55 samples and 72 genes) Supplementary Table S5. Significant copy number alterations per subtype.

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor–positive (ER+) breast cancers treated with prolonged neoadjuvant letrozole.Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER+ breast cancers from patients treated with preoperative letrozole (median, 7 months).Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P = 2.56E−15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER+ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER+ tumors in METABRIC.Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER+ breast cancer cells and in patients' ER+ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517–29. ©2018 AACR.

This file contains Supplementary Methods.

Inhibition of proliferation in estrogen receptor-positive (ER

Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post-translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR-in 32 formalin-fixed, paraffin-embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors-demonstrated significant (P0.001) overexpression of four miRNAs in epithelioid sarcomas: miR-206, miR-381, miR-671-5p, and miR-765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real-time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR-206, miR-381, and miR-671-5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR-206. Transfection of miR-206, miR-381, miR-671-5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs.

The HER2 (ERBB2) oncogene is amplified in 20-25% of breast cancers and is associated with poor patient outcome. Activating mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), occur in ∼30% of breast cancers. HER2 amplification and PIK3CA mutations often co-occur in breast cancer. Aberrant activation of the PI3K pathway correlates with a diminished response to HER2-directed therapies. We previously generated a conditional transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. We showed that PIK3CAH1047R accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers and generates resistance to FDA-approved combinations of anti-HER2 therapies (Hanker et al. PNAS 2013). HER2+/PIK3CA tumor growth was inhibited by treatment with the HER2 antibodies trastuzumab and pertuzumab in combination with the pan-PI3K inhibitor BKM120 (TPB). We sought to discover mechanisms of acquired resistance to the triple therapy by long-term treatment of established HER2+/PIK3CA tumors. We utilized tumor transplants derived from two HER2+/PIK3CA transgenic mice, #564 and #635. Tumor transplants from model 564 were initially growth inhibited by TPB, but did not regress. A subset of 564 transplants (3/11) resumed growth in the presence of continuous TPB therapy. Resistance was maintained following passaging in mice and tumors were cross-resistant to trastuzumab/pertuzumab/BYL719, a p110α-specific inhibitor. P-AKT remained suppressed in resistant tumors, whereas P-ERK was elevated. All transplants (n=9) from model 635 regressed to a volume of Citation Format: Ariella B. Hanker, Christian D. Young, Thomas P. Stricker, Rebecca S. Cook, Carlos L. Arteaga. HER2/PIK3CAH1047R transgenic tumors develop acquired resistance to triple therapy with trastuzumab, pertuzumab, and PI3K inhibitors via multiple mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1822. doi:10.1158/1538-7445.AM2014-1822