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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.

A small molecule inhibitor of MAP/ERK kinase (MEK) was effective against human breast cancer cells with a basal-like gene expression signature. Antitumor activity was limited by both feedback upregulation of phosphatidylinositol-3 kinase (PI3K)/AKT upon inhibition of MEK as well as loss of the phosphatase PTEN. Therefore, MEK inhibitors should preferably be investigated in combination with PI3K inhibitors in basal-like breast cancers.(Clin Cancer Res 2009;15(14) July 2009).

Supplementary Figure 7 - PDF file 282K, Inhibition of P-Akt in xenografts treated with trastuzumab plus/minus lapatinib

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.

Characteristics of TBCRC023 Patients with disease progression

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

Purpose:Prior neoadjuvant trials with 12 weeks of dual anti-HER2 therapy without chemotherapy demonstrated a meaningful pathologic complete response (pCR) in patients with HER2-positive breast cancer. In this trial, we sought to determine whether longer treatment would increase the rate of pCR.Patients and Methods:TBCRC023 (NCT00999804) is a randomized phase II trial combining a Simon phase II design in the experimental arm with a pick-the-winner design, not powered for direct comparison. Women with HER2-positive breast tumors measuring ≥2 cm (median = 5 cm) were randomized in a 1:2 ratio to 12 versus 24 weeks of lapatinib and trastuzumab. Letrozole (along with ovarian suppression if premenopausal) was administered in patients whose tumors were also estrogen receptor (ER) positive. All evaluable patients were assessed for in-breast pCR.Results:Ninety-seven patients were enrolled (33 in 12-week arm and 64 in 24-week arm), of whom 94 were evaluable. Median age was 51 years, and 55% were postmenopausal. Median tumor size was 5 cm, and 65% were ER-positive. The rate of pCR in the 24-week arm was 28% and numerically superior to the 12-week arm (12%). This was driven by increased pCR in the ER-positive subgroup (33% vs. 9%). Study treatment was well tolerated, with grade 1–2 diarrhea and acneiform rash being the most common toxicities.Conclusions:Treatment with dual anti-HER2 therapy for 24 weeks led to a numeric increase in pCR rate in women with HER2-positive breast cancer, without using chemotherapy. If validated, this approach may help identify patients who may benefit from deescalation of therapy.

Supplementary Figure Legends - PDF file 98K, Legends for supplementary figures 1-7

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

Supplementary Tables

HER2 overexpression drives Akt signaling and cell survival and HER2-enriched breast tumors have a poor outcome when Akt is upregulated. Akt is activated by phosphorylation at T308 via PI3K and S473 via mTORC2. The importance of PI3K-activated Akt signaling is well documented in HER2-amplified breast cancer models, but the significance of mTORC2-activated Akt signaling in this setting remains uncertain. We report here that the mTORC2 obligate cofactor Rictor is enriched in HER2-amplified samples, correlating with increased phosphorylation at S473 on Akt. In invasive breast cancer specimens, Rictor expression was upregulated significantly compared with nonmalignant tissues. In a HER2/Neu mouse model of breast cancer, genetic ablation of Rictor decreased cell survival and phosphorylation at S473 on Akt, delaying tumor latency, penetrance, and burden. In HER2-amplified cells, exposure to an mTORC1/2 dual kinase inhibitor decreased Akt-dependent cell survival, including in cells resistant to lapatinib, where cytotoxicity could be restored. We replicated these findings by silencing Rictor in breast cancer cell lines, but not silencing the mTORC1 cofactor Raptor (RPTOR). Taken together, our findings establish that Rictor/mTORC2 signaling drives Akt-dependent tumor progression in HER2-amplified breast cancers, rationalizing clinical investigation of dual mTORC1/2 kinase inhibitors and developing mTORC2-specific inhibitors for use in this setting. Cancer Res; 76(16); 4752–64. ©2016 AACR.

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

Supplementary Figure 2 - PDF file 141K, Expression of T798M HER2 results in increased HER3 phosphorylation and HER3-p85 association

Supplementary Figure 5 - PDF file 77K, HER3 expression is required for growth of T798M mutant-expressing cells

Supplementary Figure 4 - PDF file 152K, T798M mutant expression in as low as 5% of cells is sufficient to confer resistance

Related Article from Inhibition of PI3K and MEK: It Is All about Combinations and Biomarkers

Supplementary Figure 1 - PDF file 189K, Pan-ErbB inhibitor CI-1033 inhibits HER2 phosphorylation and cell growth of both wild-type and T798M mutant cells

Supplementary Figure 6 - PDF file 293K, Inhibition of P-Akt in xenografts treated with trastuzumab plus/minus cetuximab

Supplemental Figure Legends and Supplemental tables (Table 1 and table 2)

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.

Purpose: Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M–induced resistance to identify potential strategies to overcome that resistance.Experimental Design: HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis.Results: A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance.Conclusions: Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene–amplified breast cancer cells harboring T798M mutant alleles. Clin Cancer Res; 19(19); 5390–401. ©2013 AACR.

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.

Supplemental Figure S1. Kaplan meier analysis and oncoprint mapping of Luminal A/B (A) and Basal-like (B) TCGA-curated breast cancers, showing the tumors in each set with upregulation of Akt1/2/3 mRNA (red boxes) or phospho-S473 (black arrows), and the survival of these patients (red Kaplan-Meier line) versus other patients (blue Kaplan-Meier line). Log-rank test shows insignificance. Supplemental Figure S2. Rictor expression in human breast cancer. Supplemental Figure S3. Oncoprint of TCGA-curated invasive breast cancers (TCGA, Cell 2015, N = 971) assessed for RICTOR genomic alterations. Analysis shows 8% of cases (76/971) with gene amplification, overexpression, missense mutation, or deep deletion. Supplemental Figure S4. Rictor mRNA expression was measured by qRT-PCR of total RNA from mammary glands harvested from virgin female 12-week old mice. Supplemental Figure S5. Genetic ablation of Rictor decreases growth and survival of HER2-positive breast cancer cells. Supplemental Figure S6. A. Cells were cultured 6 hours with FITC-labeled Annexin V, then imaged. B. MCF10-HER2 cells (parental, Rictor, SFN, and Rictor ZFN + exogenous expression of myc-tagged Rictor) were cultured 6 h with An-nexin V-FITC. C. The percentage of cells in panel B that were labeled with Annexin V-FITC was quantitated. Averages (midlines) ± S.D. were assessed for significance using Student€™s two-tailed unpaired T-test. Supplemental Figure S7. Rictor-mediated Akt activity is necessary and sufficient for breast cancer cell survival. Supplemental Figure S8. Cells were treated with lapatinib for the time course indicated. Total cell RNA was used for qRT-PCR to measure relative RPTOR mRNA expression. Supplemental Figure S9. Cells were cultured 7 days in 10% serum plus lapatinib (1 μM) or PP242 (1 μM) or the combination of lapatinib + PP242. Cells were stained with crystal violet. Crystal violet fluorescence was scanned using the odyssey. Representative images are shown. Supplemental Figure S10. Loss of Rictor/mTORC2 sensitizes parental and resistant cells to lapatinib in vitro Supplemental Figure S11. Cells were treated 6h with PP242 (1μM) ± lapatinib (1μM) and assessed by luminescent caspase-3/7 assay.

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.

Treatment discontinuation in TBCRC023 Patients

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.

This file contains Supplementary Methods.

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

PDF file - 203K

BackgroundCyclin-dependent kinases (CDK) 4 and 6 regulate G1 to S cell cycle progression and are often altered in cancers. Abemaciclib is a selective inhibitor of CDK4 and CDK6 approved for administration on a continuous dosing schedule as monotherapy or as combination therapy with an aromatase inhibitor or fulvestrant in patients with advanced or metastatic breast cancer. This Phase 1b study evaluated the safety and tolerability, pharmacokinetics, and antitumor activity of abemaciclib in combination with endocrine therapy for metastatic breast cancer (MBC), including aromatase inhibitors (letrozole, anastrozole, or exemestane) or tamoxifen.Patients and MethodsWomen ≥18 years old with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) MBC were eligible for enrollment. Eligibility included measurable disease or non-measurable but evaluable bone disease by Response Evaluation Criteria in Solid Tumours (RECIST) v1.1, Eastern Cooperative Oncology Group performance status 0–1, and no prior chemotherapy for metastatic disease. Adverse events were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0 and tumor response were assessed by RECIST v1.1.ResultsSixty-seven patients were enrolled and received abemaciclib 200 mg every 12 hours in combination with letrozole (Part A, n=20), anastrozole (Part B, n=16), tamoxifen (Part C, n=16), or exemestane (Part D, n=15). The most common treatment-emergent adverse events (TEAE) were diarrhea, fatigue, nausea, and abdominal pain. Grade 4 TEAEs were reported in five patients (one each with hyperglycemia, hypertension, neutropenia, procedural hemorrhage, and sepsis). There was no effect of abemaciclib or endocrine therapy on the pharmacokinetics of any combination study drug. Across all treated patients, the median progression-free survival was 25.4 months (95% confidence interval: 18.0, 35.8). The objective response rate was 38.9% in 36 patients with measurable disease.ConclusionsAbemaciclib in combination with multiple endocrine therapy options exhibited manageable safety and promising antitumor activity in patients with HR+, HER2- MBC.Clinical Trial Registrationhttps://clinicaltrials.gov/, identifier NCT02057133