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Supplementary Tables - PDF file 148K, Table S1. Characteristics of HER2-negative breast cancers, separated by HR status, TCGA Table S2. Characteristics of triple-negative breast cancers with residual disease post neoadjuvant chemotherapy and univariate Table S3. Bimodal analysis of metabolism-related genes ranked by bimodality index (BI)

Purpose: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations.Experimental Design: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER+/HER2−) and, as controls, 115 ER-negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes.Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%–21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%–41%). These mutations were not detected in primary or treatment-naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments.Conclusions: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER+ breast cancer. Clin Cancer Res; 20(7); 1757–67. ©2014 AACR.

Purpose: Although most patients with estrogen receptor α (ER)-positive breast cancer initially respond to endocrine therapy, many ultimately develop resistance to antiestrogens. However, mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped.Experimental Design: We adapted four ER+ human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy.Results: The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole, each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally, knockdown of MYC inhibited LTED cell growth.Conclusions: A gene expression signature derived from ER+ breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance. Clin Cancer Res; 17(7); 2024–34. ©2011 AACR.

Supplementary Figure 5: The change in sTILs during NAC is not prognostic for outcome in TNBC patients with residual disease.

Supplementary Figure 16: 8 gene score is predominately expressed by CD8+ T cells and NK cells in both patients.

Dataset 1: Metadata and clonotype data for TCRβ sequencing (Adaptive) in 15 pairs of NAC-treated patients (tumor data).

Supplementary Figure 2: Pre-NAC sTILs have minimal prognostic value in breast cancer patients with residual disease.

Efferocytosis is the process by which apoptotic cells are cleared from tissue by phagocytic cells. The removal of apoptotic cells prevents them from undergoing secondary necrosis and releasing their inflammation-inducing intracellular contents. Efferocytosis also limits tissue damage by increasing immunosuppressive cytokines and leukocytes and maintains tissue homeostasis by promoting tolerance to antigens derived from apoptotic cells. Thus, tumor cell efferocytosis following cytotoxic cancer treatment could impart tolerance to tumor cells evading treatment-induced apoptosis with deleterious consequences in tumor residual disease. We report here that efferocytosis cleared apoptotic tumor cells in residual disease of lapatinib-treated HER2+ mammary tumors in MMTV-Neu mice, increased immunosuppressive cytokines, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). Blockade of efferocytosis induced secondary necrosis of apoptotic cells, but failed to prevent increased tumor MDSCs, Treg, and immunosuppressive cytokines. We found that efferocytosis stimulated expression of IFN-γ, which stimulated the expression of indoleamine-2,3-dioxegenase (IDO) 1, an immune regulator known for driving maternal-fetal antigen tolerance. Combined inhibition of efferocytosis and IDO1 in tumor residual disease decreased apoptotic cell- and necrotic cell-induced immunosuppressive phenotypes, blocked tumor metastasis, and caused tumor regression in 60% of MMTV-Neu mice. This suggests that apoptotic and necrotic tumor cells, via efferocytosis and IDO1, respectively, promote tumor ‘homeostasis’ and progression.Significance:These findings show in a model of HER2+ breast cancer that necrosis secondary to impaired efferocytosis activates IDO1 to drive immunosuppression and tumor progression.

Supplemental Figure S6. Tumors harvested on treatment d 7 were assessed by IHC for FoxP3 and Arg-1. Representative images are shown. Original magnification = 400X. Asterisks represent areas of acellular debris. Black arrows indicate tumor infiltrating lymphocytes (TILs). Yellow arrows indicate hyper-condensed nuclei characteristic of apoptotic bodies / apoptotic debris.

Supplementary Tables S1-S10.

Purpose:The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).Experimental Design:We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood.Results:In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.Conclusions:We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Supplementary Figure 7: Changes in immunologic signatures in response to NAC are not associated with outcome in non-TNBC tumors with residual disease.

Supplementary Figures - PDF file 440K, Figure S1. Figure S1. Validation the IHC protocol for LDHB using FFPE MDAMB231 cells with stable knockdown of LDHA or LDHB. Reduced staining for LDHB was detected only in the LDHB knockdown line. Control cells were infected with a non-silencing shRNA. Figure S2. LDHB, when present, substantially contributed to the total LDH activity in cell lines. Figure S3. A, verification of LDHA or LDHB knockdown by LDH activity in stable isogenic cell lines, MDAMB231 and HCC1937. Figure S4. LDHB association with PAM50 intrinsic subtype in primary breast cancers. A, LDHB mRNA expression separated by intrinsic subtype: Perou GSE10893 (8) Figure S5. High LDHB was associated with triple-negative breast cancer. LDHB mRNA expression of HER2 (by IHC/FISH)-negative disease separated by clinical classification Figure S6. LDHB mRNA expression within PAM50 intrinsic subtypes for HR-positive/HER2-negative breast cancers. Figure S7. LDHB mRNA expression within PAM50 intrinsic subtypes for triple-negative breast cancers Figure S8. LDHB mRNA expression was highly associated with cell cycle proliferation marker, CCNB1, for basal-like cancers within the triple-negative group. Figure S9. Association between mRNA expression and copy number for LDHB for the breast invasive carcinoma cohort separated by (A) basal and (B) luminal A/B subtypes for TCGA "manuscript samples" within the cBio Cancer Genomics Portal

Purpose: We describe herein a novel P447_L455 deletion in the C2 domain of PIK3CA in a patient with an ER+ breast cancer with an excellent response to the PI3Kα inhibitor alpelisib. Although PIK3CA deletions are relatively rare, a significant portion of deletions cluster within amino acids 446–460 of the C2 domain, suggesting these residues are critical for p110α function.Experimental Design: A computational structural model of PIK3CAdelP447-L455 in complex with the p85 regulatory subunit and MCF10A cells expressing PIK3CAdelP447-L455 and PIK3CAH450_P458del were used to understand the phenotype of C2 domain deletions.Results: Computational modeling revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Coimmunoprecipitation experiments showed reduced binding of the C2 deletion mutants with p85 compared with wild-type p110α. The MCF10A cells expressing PIK3CA C2 deletions exhibited growth factor–independent growth, an invasive phenotype, and higher phosphorylation of AKT, ERK, and S6 compared with parental MCF10A cells. All these changes were ablated by alpelisib treatment.Conclusions: C2 domain deletions in PIK3CA generate PI3K dependence and should be considered biomarkers of sensitivity to PI3K inhibitors. Clin Cancer Res; 24(6); 1426–35. ©2017 AACR.

Supp Figure S1-S5 with legends, Supp Tables S1-S4

Supplementary Figure 3: The prognostic value of sTILs is primarily confined to the post-NAC specimen in TNBC patients with residual disease.

Supplementary Data - PDF file 89K, Supplementary Methods Tissue microarray (TMA) of triple-negative breast cancers

Supplementary Figure 6: Neither time of sample nor institution drive significant gene expression changes.

Supplementary Tables

Supplementary Figure 4: sTILs are not prognostic in ER+ or HER2+ disease.

Purpose: Although breast cancers are known to be molecularly heterogeneous, their metabolic phenotype is less well-understood and may predict response to chemotherapy. This study aimed to evaluate metabolic genes as individual predictive biomarkers in breast cancer.Experimental Design: mRNA microarray data from breast cancer cell lines were used to identify bimodal genes—those with highest potential for robust high/low classification in clinical assays. Metabolic function was evaluated in vitro for the highest scoring metabolic gene, lactate dehydrogenase B (LDHB). Its expression was associated with neoadjuvant chemotherapy response and relapse within clinical and PAM50-derived subtypes.Results: LDHB was highly expressed in cell lines with glycolytic, basal-like phenotypes. Stable knockdown of LDHB in cell lines reduced glycolytic dependence, linking LDHB expression directly to metabolic function. Using patient datasets, LDHB was highly expressed in basal-like cancers and could predict basal-like subtype within clinical groups [OR = 21 for hormone receptor (HR)-positive/HER2-negative; OR = 10 for triple-negative]. Furthermore, high LDHB predicted pathologic complete response (pCR) to neoadjuvant chemotherapy for both HR-positive/HER2-negative (OR = 4.1, P < 0.001) and triple-negative (OR = 3.0, P = 0.003) cancers. For triple-negative tumors without pCR, high LDHB posttreatment also identified proliferative tumors with increased risk of recurrence (HR = 2.2, P = 0.006).Conclusions: Expression of LDHB predicted response to neoadjuvant chemotherapy within clinical subtypes independently of standard prognostic markers and PAM50 subtyping. These observations support prospective clinical evaluation of LDHB as a predictive marker of response for patients with breast cancer receiving neoadjuvant chemotherapy. Clin Cancer Res; 19(13); 3703–13. ©2013 AACR.

PDF file - 187K, Supplementary Table 1: Detailed demographic information of all patients with metastatic breast cancer, including cohorts LM+ and EM+. Supplementary Table 2: ESR1 variant sequence data details. Abbreviations: cvg, coverage, N/A, non-applicable. Supplementary Table 3: List of genes sequenced by captured next generation sequencing. Supplementary figure 1: Genomic profiles of primary and metastatic tumors. Genomic alterations were found in 32 genes in primary and metastatic ER+ breast cancers. Genes are listed from the most frequently altered to least altered gene. Supplementary figure 2: No change in WT and mutant ER activity across a wide range of E2 doses. Luciferase activity in 293T cells after co-transfection of the ERE-TK-Luc reporter vector along with WT-ER, Y537N or D538G and E2 stimulation using doses of E2 ranging from 0.01nM to 100nM or vehicle treatment.

Supplemental Figure 1: No clear interaction of molecular subtype with change in TIL score dureing NAC. Supplemental Figure 2: No clear relationship between pre-NAC TILs or change in TILs with RFS or OS. Supplemental Figure 3: No clear association between pre-NAC or post-NAC TILs and total number of targeted alterations identified by targeted NGS. Supplemental Figure 4: A) Percent of high expressers of PD-L1 (>1 standard deviation from the mean) in each molecular subtype in the TCGA breast cancer dataset. Supplemental Figure 5: MEK inhibition down-regulates downstream ERK signalling and inhibits proliferation of TNBC murine breast cancer lines AT3ova and 4T1.9 in vitro. Supplemental Figure 6: Efficacy of selumetinib and anti-PD-L1 therapy in MMTV-Neu models. Supplemental Figure 7: Pharmacodynamic activity of trametinib and selumetinib in vivo.

Purpose:The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib.Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses.Results:Mice receiving “extended adjuvant” therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2− MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells.Conclusions:These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.

Dataset 2: Metadata and clonotype data for TCRβ sequencing (Archer) in 4 patients (peripheral CD8+ PD-1HI and CD8+ PD-1NEG T cells and post-NAC tumor), before (n=3) and after (n=4) NAC.

Supplementary Figure S1; Supplementary Methods.

Purpose: Tumor-infiltrating lymphocytes (TIL) in the residual disease (RD) of triple-negative breast cancers (TNBC) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into tumor cell-autonomous molecular pathways affecting these features are lacking.Experimental Design: We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1–targeted immunotherapy in mouse models of breast cancer.Results: Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras–MAPK signaling were significantly correlated with lower TILs. MEK inhibition upregulated cell surface MHC expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PD-L1/PD-1 inhibition enhanced antitumor immune responses in mouse models of breast cancer.Conclusions: These data suggest the possibility that Ras–MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1–targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors. Clin Cancer Res; 22(6); 1499–509. ©2015 AACR.

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Supplementary figure files

Supplementary figure legend

Supplementary Figure 8: Changes in immune-related genes in response to NAC are not associated with outcome in ER+ or HER2+ tumors with residual disease.

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Supplementary Methods, Figure Legends 1-13, Table 1 from A Kinome-Wide Screen Identifies the Insulin/IGF-I Receptor Pathway as a Mechanism of Escape from Hormone Dependence in Breast Cancer

Supplementary information - PDF file 2943K, Supplementary Figure 1: Dox-induced iPIK3.iCre transgenic mouse model of PIK3CA mutant breast cancer. Supplementary Figure 2: Loss of ErbB3 delays mammary gland hyperplasia at 12 weeks. Supplementary Figure 3: IVIS Bioluminescent imaging. Supplementary Figure 4: Histology of PIK3CA-induced mammary tumors remains diverse when ErbB3 is lost. Supplementary Figure 5: Mutant PI3K binds to IRS-1, Gab1 and other tyrosinephosphorylated proteins in tumors treated with lapatinib and/or imatinib. Supplementary Figure 6: Combination of BYL719 and lapatinib delays tumor growth without causing liver toxicity

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Supplementary Figure 3 from Trastuzumab Has Preferential Activity against Breast Cancers Driven by HER2 Homodimers

Supplementary Figure 5 from Trastuzumab Has Preferential Activity against Breast Cancers Driven by HER2 Homodimers

Basal-like breast cancer (BLBC) is an aggressive disease that lacks a clinically approved targeted therapy. Traditional chemotherapy is effective in BLBC, but it spares the cancer stem cell (CSC)-like population, which is likely to contribute to cancer recurrence after the initial treatment. Dual specificity phosphatase-4 (DUSP4) is a negative regulator of the mitogen-activated protein kinase (MAPK) pathway that is deficient in highly aggressive BLBCs treated with chemotherapy, leading to aberrant MAPK activation and resistance to taxane-induced apoptosis. Herein, we investigated how DUSP4 regulates the MAP–ERK kinase (MEK) and c-jun-NH2-kinase (JNK) pathways in modifying CSC-like behavior. DUSP4 loss increased mammosphere formation and the expression of the CSC-promoting cytokines interleukin (IL)-6 and IL-8. These effects were caused in part by loss of control of the MEK and JNK pathways and involved downstream activation of the ETS-1 and c-JUN transcription factors. Enforced expression of DUSP4 reduced the CD44+/CD24− population in multiple BLBC cell lines in a MEK-dependent manner, limiting tumor formation of claudin-low SUM159PT cells in mice. Our findings support the evaluation of MEK and JNK pathway inhibitors as therapeutic agents in BLBC to eliminate the CSC population. Cancer Res; 73(20); 6346–58. ©2013 AACR.

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Legends for Supplemental Figures S1-S6

Estrogen receptor α (ER)–positive breast cancers adapt to hormone deprivation and become resistant to antiestrogens. In this study, we sought to identify kinases essential for growth of ER+ breast cancer cells resistant to long-term estrogen deprivation (LTED). A kinome-wide siRNA screen showed that the insulin receptor (InsR) is required for growth of MCF-7/LTED cells. Knockdown of InsR and/or insulin-like growth factor-I receptor (IGF-IR) inhibited growth of 3 of 4 LTED cell lines. Inhibition of InsR and IGF-IR with the dual tyrosine kinase inhibitor OSI-906 prevented the emergence of hormone-independent cells and tumors in vivo, inhibited parental and LTED cell growth and PI3K/AKT signaling, and suppressed growth of established MCF-7 xenografts in ovariectomized mice, whereas treatment with the neutralizing IGF-IR monoclonal antibody MAB391 was ineffective. Combined treatment with OSI-906 and the ER downregulator fulvestrant more effectively suppressed hormone-independent tumor growth than either drug alone. Finally, an insulin/IGF-I gene expression signature predicted recurrence-free survival in patients with ER+ breast cancer treated with the antiestrogen tamoxifen. We conclude that therapeutic targeting of both InsR and IGF-IR should be more effective than targeting IGF-IR alone in abrogating resistance to endocrine therapy in breast cancer. Cancer Res; 71(21); 6773–84. ©2011 AACR.

Supplementary Figure S5. PLK1 targeting decreases the expression of ER signaling genes.

Description of additional methods and procedures used in the study.

PDF file, 392K, Correlation of DUSP4 gene expression and DUSP4 copy number across 444 breast cancer samples (S1); Cytokine array of conditioned media from MDA-231 cells after DUSP4 knockdown (S2); Upregulation of IL6 and IL8 after sustained shDUSP4 expression in MDA-231 cells (S3); MEK pathway inhibition limits mammosphere formation and self-renewal in MDA231 cells (S4); Association of genomic DUSP4 loss with p-cJUN expression in the TCGA data (S5); Chromatin Immunoprecipitation of ETS-1 at the IL8 promoter (S6); Mammosphere quantification of SUM159PT cells in the presence of alternative MEK inhibitors (S7); Inhibition of MEK and JNK increases the CD24+ population in BLBC cells (S8); Escape from long term enforced expression of DUSP4 in SUM159PT/pINDUCER-DUSP4 cells (S9).

Supplementary Figure 2 from Trastuzumab Has Preferential Activity against Breast Cancers Driven by HER2 Homodimers

Supplementary Table S2. siRNAs identified to decrease cell proliferation, ER transcription and both in the RNAi screen of MCF7/LTED cells.

Supplementary Table and Figure Legends. Legends for Supplementary Tables S1-S2 and Supplementary Figures S1-S6.

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

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