Search

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

Background:Increased body mass index (BMI) and metabolic syndrome are associated with an increased risk of breast cancer recurrence. The mechanism(s) responsible for this increased risk are not known, though multiple mechanisms including resistance to endocrine therapy have been proposed. We sought to determine whether obesity and/or metabolic syndrome were associated with intrinsic tumor risk as determined by the Oncotype recurrence score (RS), or with resistance to neoadjuvant endocrine therapy, as determined by Ki67 response after short-term presurgical treatment. Methods:To investigate whether BMI was associated with increased intrinsic recurrence risk, we analyzed a cohort of 779 patients treated at Vanderbilt for early stage breast cancer and who had Oncotype test results. Medical records were reviewed to record BMI at the time of breast cancer diagnosis. We compared the Oncotype RS between patients with BMI greater or less than 27, and in normal, overweight, and obese ranges. To investigate whether the increase in breast cancer recurrence risk associated with obesity could be identified by a surrogate marker of short-term response to endocrine therapy, we analyzed data from a presurgical trial of endocrine therapy in early stage breast cancer patients previously completed at Vanderbilt. In this study patients were treated with 1-2 weeks of letrozole prior to surgery, then the surgical specimen was analyzed for Ki67. The original goal of the study was to compare endocrine sensitive tumors with endocrine resistant tumors as a platform for discovery of resistance mechanisms to endocrine therapy. We retrospectively analyzed medical records for 143 patients in that study who had post-treatment Ki67 values available, and for whom BMI at the time of surgery could be obtained. We also reviewed medical records for components of the metabolic syndrome (hypertension, hyperglycemia or anti-hyperglycemic medications, low HDL, high triglycerides or statin use, and BMI > 30 as a surrogate for waist circumference). Metabolic syndrome was defined as 3 or more of the 5 components. For endocrine therapy response, a cutoff of 1 for the natural log Ki67 expression was defined as sensitive (approximately 2.5%) and tumors with natural log of Ki67 expression between 1 and 2 (approximately 7.5%) were categorized as intermediate sensitivity. Results:We did not find any significant association between BMI and recurrence score and the distribution of risk scores was similar across all BMI groups. We did not observe any correlation between RS and BMI. However we did observe a difference in the proportion of endocrine sensitive and resistant tumors in overweight and obese groups (only a minority of patients in this cohort, 18%, had normal weight). In patients with overweight, 70% of the tumors had a post-treatment Ki67 level classified as sensitive, whereas only 40% of the tumors in patients with obesity were endocrine sensitive (chi square = 0.0518). Similarly, 72% of patients without metabolic syndrome had sensitive tumors, but only 51% of patients with metabolic syndrome had sensitive tumors (relative risk 1.408, chi square = 0.0154). Conclusions:Our findings suggest that the association between obesity and/or metabolic syndrome and increased recurrence risk is not reflected in tumor-intrinsic properties present at the time of diagnosis. Rather, the effect of obesity and metabolic syndrome appears to be in response to treatment, at least as measured by a short-term surrogate marker of long-term endocrine sensitivity. Importantly, this suggests that interventions to reverse obesity and/or metabolic syndrome may be effective in improving outcomes for breast cancer recurrence, at least in part by improving sensitivity to endocrine therapy. Citation Format: Riley Bergman, Yvonne Berko, Brent Rexer. Association of factors of the obesity phenotype with breast cancer recurrence risk measured by oncotype recurrence score and short-term response to neoadjuvant endocrine therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-18.

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.

The recent approval of anti-PD-L1 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). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-15.

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

Background: Somatic alterations in the FGFR pathway (mainly FGFR1, amplified in about 15% of ER+ BC) have been implicated in resistance to endocrine therapy (ET), and more recently, to CDK4/6i as well. Based on our preclinical data showing that the FGFR pan-inhibitor erdafitinib when added to fulvestrant/palbociclib resulted in marked PDX regressions, we initiated a phase Ib trial combining erdafitinib with fulvestrant/palbociclib in patients with ER+/HER2-/FGFR-amplified MBC (NCT03238196) to determine safety, tolerability and anti-tumor activity of this combination. Methods: Patients with evaluable ER+/HER2- MBC with FGFR1-4 amplification (detected on tumor next generation sequencing or plasma ctDNA) exposed to at least one ET regimen (but no more than 2 lines of chemotherapy) in the metastatic setting, were treated with fulvestrant, palbociclib (standard of care dosing/ schedule) and oral erdafitinib, tested in 4 doses ranging from 4 mg to 8 mg daily. Once MTD reached, we planned to enroll 20 patients in the expansion portion of the trial. Tumor blocks were collected for FGFR1 FISH amplification analysis, and plasma ctDNA was collected at baseline, 4 weeks and at treatment discontinuation. Tumor assessments were performed every 8 weeks. Results: Since August 2017, 26 eligible patients with evaluable ER+/HER2-/FGFR-amplified MBC were enrolled across 4 institutions. Patient characteristics are summarized in Table 1. Main grade 1 and 2 adverse events (AE) were consistent with on-target toxicities of erdafitinib and/or palbociclib: mucositis (67%), hyperphosphatemia (61%), dysgeusia (52%), diarrhea (48%), fatigue (48%), neutropenia (47%), hand-foot syndrome (38%), anemia (29%), and onycholysis (14%). Febrile neutropenia occurred in 5% patients, no cases of central serous retinopathy were seen. Serious AE were rare: one grade 4 elevation of transaminases (DLT; attributed to fulvestrant), one grade 3 colitis (attributed to erdafitinib), and one thromboembolic event (attributed to palbociclib). In combination with fulvestrant/ palbociclib, the MTD of erdafitinib was 6 mg. No drug-drug interaction was seen. 8 patients were deemed non-evaluable for anti-tumor effect as treatment discontinuation (mainly due to AE) occurred prior to first tumor assessment. Of the 18 evaluable patients: 7 had disease progression, 8 had stable disease (4 of which discontinued treatment due to AE), 3 have not completed their first tumor assessment, 4 are still on treatment; median PFS was 3 months and CBR at 6 months was 28%. However, higher PFS (6 months) was seen in 6/8 patients with high levels of FGFR1 amplification (FISH FGFR1:CEP8 ratio >5; gene copy number >10) and in both patients with FGFR3 amplification. Conclusion: To our knowledge, this is the first time an FGFR inhibitor has been tested in combination with ET and CDK4/6i in patients with MBC harboring FGFR alterations. Erdafitinib-related side effects appeared to be on target, leading to treatment discontinuation in several patients despite optimal medical treatment. Clinical activity was seen in heavily pre-treated patients with molecular evidence of high FGFR amplification despite 100% prior exposure to ET and CDK4/6i. Full clinical and correlative work will be presented at the meeting, and a future phase II trial is being planned. Table 126 / 35 patients accrued13 in escalation, 13 in expansion(ongoing)Median age53 (35 - 75)Race/ ethnicityWhite 22Black 1Asian 2Hispanic 1Median number of lines of treatment in the metastatic setting4 (1 - 5)Prior lines of treatment in the metastatic settingEndocrine therapy 100%Fulvestrant 28%CDK4/6i 100%PI3K pathway inhibitor 80%1 line chemo 65%2 lines chemo 45%FGFR1 amplification23FGFR3 amplification2FGFR4 amplification1 Citation Format: Ingrid A. Mayer, Barbara B. Haley, Vandana G. Abramson, Adam Brufsky, Brent Rexer, Erica Stringer-Reasor, Komal L. Jhaveri, Melinda Sanders, Paula I. Ericsson-Gonzalez, Fei Ye, Carlos L. Arteaga. A phase Ib trial of fulvestrant + CDK4/6 inhibitor (CDK4/6i) palbociclib + pan-FGFR tyrosine kinase inhibitor (TKI) erdafitinib in FGFR-amplified/ ER+/ HER2-negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-03.

Background Identification of HER2-positive breast cancers with high anti-HER2 sensitivity could help de-escalate chemotherapy. Here, we tested a clinically applicable RNA-based assay that combines ERBB2 and the HER2-enriched (HER2-E) intrinsic subtype in HER2-positive disease treated with dual HER2-blockade without chemotherapy. Methods A research-based PAM50 assay was applied in 422 HER2-positive tumors from five II–III clinical trials (SOLTI-PAMELA, TBCRC023, TBCRC006, PER-ELISA, EGF104090). In SOLTI-PAMELA, TBCRC023, TBCRC006, and PER-ELISA, all patients had early disease and were treated with neoadjuvant lapatinib or pertuzumab plus trastuzumab for 12–24 weeks. Primary outcome was pathological complete response (pCR). In EGF104900, 296 women with advanced disease were randomized to receive either lapatinib alone or lapatinib plus trastuzumab. Progression-free survival (PFS), overall response rate (ORR), and overall survival (OS) were evaluated. Results A total of 305 patients with early and 117 patients with advanced HER2-positive disease were analyzed. In early disease, HER2-E represented 83.8% and 44.7% of ERBB2-high and ERBB2-low tumors, respectively. Following lapatinib and trastuzumab, the HER2-E and ERBB2 (HER2-E/ERBB2)-high group showed a higher pCR rate compared to the rest (44.5%, 95% confidence interval [CI] = 35.4% to 53.9% vs 11.6%, 95% CI = 6.9% to 18.0%; adjusted odds ratio [OR] = 6.05, 95% CI = 3.10 to 11.80, P Conclusions Combining HER2-E subtype and ERBB2 mRNA into a single assay identifies tumors with high responsiveness to HER2-targeted therapy. This biomarker could help de-escalate chemotherapy in approximately 40% of patients with HER2-positive breast cancer.

Background: HER2-positive breast cancer consists of 4 intrinsic molecular subtypes (Luminal A, Luminal B, HER2-enriched [HER2-E], and Basal-like) and a Normal-like subtype, with the HER2-E subtype having the highest activation of the EGFR-HER2 pathway. Concordant with this, the HER2-E subtype was significantly associated with pathological complete response (pCR) following lapatinib and trastuzumab without chemotherapy on the PAMELA phase II neoadjuvant trial (Lancet Oncol 2017). Here, we aimed to further validate this observation in a different cohort using tumor samples from the TBCRC023 trial. Methods: TBCRC023 (NCT00999804) was a randomized phase II trial combining a Simon Phase 2 design in the experimental arm with a pick-the-winner design, not powered for direct comparison. Ninety-seven women with HER2+ breast cancer measuring 2 cm or larger (median = 5 cm) were randomized in a 1:2 ratio to 12 vs. 24 weeks of lapatinib and trastuzumab. Letrozole (along with ovarian suppression if premenopausal) was added in patients whose tumors were also estrogen receptor (ER)-positive. All evaluable patients were assessed for pCR, defined as no residual invasive carcinoma in the breast. Intrinsic molecular subtypes from tumor biopsy formalin-fixed, paraffin-embedded samples taken at baseline (day 0) were determined with the nCounter-based PAM50 predictor. Gene expression PAM50 data was performed at Hospital Clínic in Barcelona blinded from clinical data. The primary outcome was the association of the HER2-E subtype (vs. non-HER2-E) with pCR. A logistic regression model adjusted for tumor size, ER status, nodal status and treatment arm was performed. Results: A total of 85 of the 97 (87.6%) baseline tumor samples were available. Most patients had the HER2-E subtype (51 [60.0%]), followed by Normal-like (12 [14.1%]), Basal-like (11 [13.0%]), Luminal B (7 [8.2%]) and Luminal A (4 [4.7%]). The proportion of patients with HER2-E tumors within ER+ and ER-negative disease was 54.9% and 67.7%, respectively. At the time of surgery, 17 of 85 patients (20.0%; 95% confidence interval [CI] 0.13-0.30) had a pCR in the breast. Fourteen of 51 patients with the HER2-E subtype (27.5%; 95% CI 0.17 to 0.41) and 3 of 34 patients with non-HER2-E subtypes (8.8%; 95% CI 0.03 to 0.23) achieved a pCR at the time of surgery (adjusted odds ratio 4.33; 95% CI 1.08-17.41; P=0.039). No other clinical-pathological variable was found significantly associated with pCR in the multivariable model. Conclusions: In an independent validation study performed while blinded to clinical outcomes, HER2-E subtype confirms its ability to identify patients with HER2-positive breast cancer who are likely to benefit from dual HER2 blockade therapies without chemotherapy. Further studies should be performed to prospectively validate this biomarker, alone or in combination with other biomarkers. Citation Format: Prat A, De Angelis C, Pascual T, Gutierrez C, Wang T, Paré L, Rexer B, Cortes J, Forero A, Llombart A, Wolff AC, Krop I, Galván P, Pavlick AC, Villagrasa P, Hilsenbeck SG, Schiff R, Osborne CK, Rimawi MF. Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-12.

Background: FGFR1 amplification (amp) occurs in ˜15% of breast cancers (BC) and associates with poor prognosis and resistance to endocrine therapy. CCND1 regulates cell cycle progression and is amplified in 15-20% of BC. Co-amp of FGFR1 occurs in 30-40% of CCND1amp tumors, suggesting the possibility of oncogene cooperativity. CDK4/6 inhibitors, which block the action of cyclin D1/CDK4 complexes at the G1-to-S transition, are approved for treatment of ER+ BC and FGFR inhibitors are in early phase clinical trials. Results: Between 11/2013 and 03/2017, 191 BCs from 188 patients with metastatic (M) or refractory locoregional recurrent (RLRR) BC at Vanderbilt (VICC) were profiled by targeted next gen sequencing (Foundation OneTM). These are included within the 2131 publicly available BC sequencing results in GENIE (Foundation OneTM and MSK-IMPACT). Among the GENIE cohort, rates were: FGFR1amp 7% (n=156), CCND1amp 12% (n=261) and CCND1/FGFR1co-amp 3% (n=58). Additional cases showed FGFR1 missense mutations (n=16) and deep deletions (n=5). When the analysis was limited to the VICC cohort allowing restriction to ER+ BC, FGFR1amp (16%) and CCND1amp (23%) rates are similar to rates in primary BC in TCGA (13% FGFR1 [p = 0.44] and 19% CCND1 [p = 0.24]). In GENIE, the most frequent co-mutations in FGFR1amp tumors were TP53 (31%), PIK3CA (21%), GATA3 (13%), CDH1 (11%) and MAP3K1 (10%). However, TP53 and PIK3CA mutations were less common among FGFR1amp tumors than FGFR1non-amp cases (p 0.016). Histopathologic correlation on tumors from our institution show a majority of FGFR1 and/or CCND1 amp BC (64%) were ER+/HER2–; 33% of ER+/FGFR1amp tumors were PR–. Distinctive histologic features associated with FGFR1 and/or CCND1 amp were lobular histology (17%) and neuroendocrine differentiation (14%), 0-10%TILs (94%) and high proliferative rate (46%). Conclusion: FGFR1amp and CCND1amp rates in TCGA are similar to those seen in MBC/LRRBC (GENIE) suggesting FGFR1 can function as both a driver mutation and de novo mechanism of endocrine resistance early in tumorigenesis. Frequent co-amp with CCND1 and lower rates of TP53 and PIK3CAmut also support a driver role for FGFR1amp and FGFR1/CCND1co-amp. The observation of neuroendocrine features in a subset of these tumors suggests lineage plasticity. This may be a consequence of genomic alterations promoting anti-estrogen resistance and is consistent with recently published BC outcome data associating neuroendocrine differentiation with higher grade ER+ tumors, frequent 8p amp, which includes FGFR1, and worse disease-free and overall survival. The frequency of FGFR1amp suggests genotype specific trials with FGFR inhibitors would be highly feasible. Whether FGFR1/CCND1 co-amplified tumors are candidates for treatment with a combination of FGFR and CDK4/6 inhibitors requires further investigation. Citation Format: Gonzalez-Ericsson PI, Estrada MV, Formisano L, Jansen VM, Mayer IA, Rexer BN, Abramson VG, Levy M, Balko JM, Stricker TP, Arteaga CL, Sanders ME. Genenomic landscape of breast cancers with FGFR1 amplification and FGFR1/CCND1 co-amplification revealed by targeted capture next generation sequencing [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-07.

Background In a randomized trial (CREATE-X) patients who had residual disease following standard neoadjuvant chemotherapy had improved survival with the addition of adjuvant capecitabine. For patients who required radiation (RT), capecitabine was given sequentially. Concurrent capecitabine-RT may be more efficacious. We hypothesized that the safety, feasibility, and toxicity of adjuvant capecitabine-RT is not significantly different compared to adjuvant RT alone. Patient and Methods We retrospectively studied patients with stage I-III invasive mammary carcinoma. Patients treated with capecitabine-RT were matched 1:3 with control patients treated with RT alone. Logistic regression evaluated predictors of radiation dermatitis. Results A total of 64 patients were enrolled including 16 patients treated by capecitabine-RT and 48 treated by RT alone. Cohorts were balanced with regard to clinicopathologic factors. No treatment in either cohort resulted in hospitalization, short-term disability, or fatality. Most toxicities of capecitabine-RT were related to radiation dermatitis. Radiation dermatitis was not significantly different between the capecitabine-RT and RT cohort at either the grade 2 level (OR 1.36, 95% CI 0.38-4.93, p=0.63) or grade 3 level (OR 3.00, 95% CI 0.85-10.63, p=0.09) or after multivariable analysis. However, patients treated with capecitabine-RT were more likely to require modifications in the radiation schedule, including treatment breaks or cancelled fractions (44% versus 17%, OR 3.89, 95% CI 1.12-13.52, p=0.03). Conclusion Capecitabine-RT appears to be safe in the adjuvant treatment of breast cancer with comparable toxicity to RT alone. It may lead to more treatment adjustments. Prospective studies are needed to evaluate the safety and tolerability of this combination.

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.

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.

Background: Abemaciclib, a small molecule inhibitor of CDK4 and CDK6, induces G1 cell cycle arrest in Rb-proficient human cancers.1 The clinical safety profile of abemaciclib enables continuous oral dosing to achieve sustained target inhibition, resulting in single-agent antitumor activity against multiple human cancers. The drug also reaches relevant concentrations in the central nervous system and, in patients taking the drug orally, can be detected in the cerebrospinal fluid.2 For women with previously treated hormone receptor positive (HR+) metastatic breast cancer (MBC), abemaciclib as a single agent achieved a six-month clinical benefit rate of 61.1% and an objective response rate of 33.3%.3 Clinical trials investigating abemaciclib combined with fulvestrant4 or aromatase inhibitors5 have led to randomized Phase 3 studies for women with HR+ breast cancer.6,7 Methods: This Phase 1b study (NCT02057133) with multiple cohorts evaluates safety and tolerability of abemaciclib combined with endocrine or HER2-targeted therapies for MBC. Secondary objectives include pharmacokinetics (PK) and antitumor activity of abemaciclib when given in combination with other therapies. Cohorts were opened to enrollment sequentially. Patients with HR+ HER2 negative MBC received abemaciclib orally every 12 hours (Q12H) in combination with the following standard therapies daily until progression: letrozole (Part A), anastrozole (Part B), tamoxifen (Part C), exemestane (Part D), or exemestane plus everolimus (Part E). Patients with HER2 positive MBC received abemaciclib orally Q12H in combination with trastuzumab every 21 days until progression (Part F). Adverse events (AEs) were graded by NCI CTCAE v4.0 and tumor response was assessed radiographically using RECIST v1.1. Results: Abemaciclib has been combined with multiple targeted therapies for the treatment of women with MBC. We previously reported safety and early efficacy results for the combinations of abemaciclib with letrozole, anastrozole, tamoxifen, exemestane, and exemestane plus everolimus.5 Due to limited follow-up at that time, the efficacy results were not mature. Safety, PK, and efficacy results with approximately 6 months of additional follow-up will be reported across all parts of the study. The most common treatment-emergent AEs include effects on the gastrointestinal and hematopoietic systems. Consistent with previously reported results for both single-agent abemaciclib and the combination of abemaciclib with fulvestrant, tumor responses have been observed among women receiving abemaciclib in combination with targeted therapies for MBC. Conclusions: This study for women with MBC demonstrates the potential for abemaciclib to be combined with therapies targeting specific signaling pathways. References: 1Gelbert et al. Invest New Drugs. 2014;32(5):825-37. 2Shapiro et al. J Clin Oncol 31, 2013 (suppl; abstr 2500). 3Tolaney et al. SABCS 2014: Abstract 763. 4Patnaik et al. J Clin Oncol 32:5s, 2014 (suppl; abstr 534). 5Tolaney et al. J Clin Oncol 33, 2015 (suppl; abstr 522). 6Llombart et al. SABCS 2014: OT1-1-07 (MONARCH 2, NCT02107703). 7Goetz et al. J Clin Oncol 33, 2015 (suppl; abstr TPS624) (MONARCH 3, NCT02246621). Citation Format: Goetz MP, Beeram M, Beck T, Conlin AK, Dees EC, Dickler MN, Helsten TL, Conkling PR, Edenfield WJ, Richards DA, Turner PK, Cai N, Chan EM, Pant S, Becerra CH, Kalinsky K, Puhalla SL, Rexer BN, Burris HA, Tolaney SM. Abemaciclib, an inhibitor of CDK4 and CDK6, combined with endocrine and HER2-targeted therapies for women with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-25.

1011 Background: Several trials have shown 25-30% pathologic complete response (pCR) rates in patients with HER2+ BC treated with LT therapy (+ endocrine therapy if ER+), but no chemotherapy (CTX). We hypothesize that a multiparameter classifier, comprised of HER2 gene and protein levels, intratumor heterogeneity (ITH), HER2-enriched (E) subtype, and PIK3CA mutation status can identify patients whose tumors are “addicted” to HER2 signaling and are likely to achieve pCR from a CTX-sparing de-escalation strategy. Methods: Baseline specimens from 2 trials (TBCRC023 [NCT00999804] and PAMELA [NCT01973660]) of neoadjuvant CTX-sparing LT (+ endocrine therapy if ER+) in HER2+ BC were used. HER2 protein and ITH (scored for % 3+ by IHC), and gene amplification (HER2:CEP17 ratio and copy number (CN) by CISH) were measured on the same slide by the dual gene protein assay (GPA). HER2-E and PIK3CA mutation status were assessed by research-based PAM50 and MSK-IMPACT platforms, respectively. A decision tree algorithm was used to determine the GPA cutoffs and to construct the classifier of response (by pCR) in TBCRC023, which was then validated in PAMELA. Results: Of the evaluable patients from TBCRC023 (N = 130) and PAMELA (N = 151), GPA data were available for 121 and 94 cases, respectively. Both cohorts exhibited similar distributions for HER2 ratio, CN, and % 3+, and a strong correlation between HER2 ratio and CN (R > 0.92). In TBCRC023, 73 cases had data from GPA, PAM50, and IMPACT, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio > 4.6 and % 3+ > 97.5% in both the GPA data cohort (N = 121) and complete data cohort (N = 73). With PAM50 and IMPACT data, the model added HER2-E and PIK3CA wild-type (wt). For practical reasons, the classifier was locked as HER2 ratio ≥ 4.5 AND % 3+ ≥ 90% AND PIK3CA-wt AND HER2-E, which yielded a PPV of 55% and NPV of 94%. Validation in PAMELA using 45 cases with data for all 3 assays yielded PPV of 44% and NPV of 82%. 29 TBCRC023 cases without IMPACT data could be predicted to be non-pCR, of which 26 were correct (NPV = 89%). In PAMELA, 66 additional cases could be predicted to be non-pCR, of which 54 were correct (NPV = 81%). Conclusions: We have constructed a multiparameter classifier that can predict pCR with targeted therapy alone that compare to pCR rates of CTX + dual anti-HER2 in unselected patients. Prospective validation in a clinical trial is warranted. [Table: see text]

Background: Current evidence indicates that patients with Human Epidermal Growth Factor Receptor 2 (HER2)-positive Breast Cancer (BC) benefit from HER2-targeted therapies. Accurate determination of HER2 status is critical to ensure that the correct patients are offered targeted treatment while patients with HER2-negative tumors–who are unlikely to benefit from anti-HER2 therapy– are spared from the adverse effects of these costly drugs. Guidelines for performance and reporting of HER2 testing, first published in 2007 by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP), were updated in October 2013. The new reporting criteria are based on a combination of HER2:CEP17 ratio and average HER2 copy number. Methods: We retrospectively reviewed HER2 dual probe FISH test results sequentially performed by the Cytogenetics Laboratory at Vanderbilt University from January to May 2014 since implementation of the updated guidelines. The cases were then rescored using the 2007 guidelines. The clinicopathologic features of cases with a change in Her2 status after scoring with the 2013 guidelines were examined for statistical significance. Results: If the 266 performed HER2 FISH tests had been scored using the 2007 guidelines the results would have been the following: amplified (n = 44, 16%), equivocal (n = 28, 10%) and not amplified (n = 194, 70%). However, using the 2013 guidelines the cases were actually reported as follows: amplified (n=57, 21%), equivocal (n=22, 8.3%) and not amplified (n=187, 68%). Additionally, 18 cases demonstrated genetic heterogeneity in >25% of cells. The updated guidelines resulted in a change in Her2 status in 12% of tests (32/266; p less than 0.0001); 13 changed from equivocal to amplified, 13 cases changed from not amplified to equivocal and 6 cases changed from equivocal to not amplified. Cases with a change in Her2 status following implementation of the new guidelines were more likely to demonstrate genetic heterogeneity (28% [9/32] vs. 4% [9/234]; p less than 0.0001). Furthermore, hormone receptor (HR)-negative tumors scored as not amplified by the 2007 guidelines were more likely to undergo an upgrade in HER2 status under the 2013 guidelines than HR-positive tumors (26% [11/41] vs. 7% [11/153], p less than 0.0001). Conclusions: The 2013 reporting criteria, based on a combination of HER2:CEP17 ratio and average HER2 copy number, increase the number of patients eligible for HER2-targeted therapies while decreasing equivocal results. Tumors that demonstrate genetic heterogeneity for Her2 or are HR-negative account for a significant percentage of these newly eligible cases. Correlation with clinical response is required to confirm the proposed improved analytical validity of the updated guidelines. Clinical trials to evaluate the benefit of anti-Her2 therapy in patients with genetic heterogeneity are in the planning phase. Citation Format: Monica V Estrada, Jena M Giltnane, Ferrin C Wheeler, Ashwini Yenamandra, Vandana Abramson, Ingrid A Mayer, Julie Means, Brent Rexer, Ingrid M Meszoely, Melinda E Sanders. Genetic heterogeneity for Her2 accounts for a significant percentage of breast cancers changing Her2 status following implementation of the 2013 CAP/ASCO HER2 reporting guidelines [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-32.