Boughey, Judy C.

Abstract INTRODUCTION Next generation sequencing (NGS) of patients has significantly changed our ways to study cancer genomics as it provides precise estimates of gene expression, fusion transcripts, expressed single nucleotide variants (eSNVs), splice variants and copy number variants. The Breast Cancer Genome Guided Therapy (BEAUTY) is an ongoing clinical study in which RNA sequencing (RNAseq) and whole exome sequencing (WES) are performed prior to, during and after neoadjuvant chemotherapy. Here we report the use of these sequencing technologies to investigate gene expression levels and mutational profiles in a triple negative breast cancer (TNBC) patient enrolled in BEAUTY whose disease did not respond to neoadjuvant paclitaxel and anthracycline/cycphosphamide. METHODS Computational approaches were used to integrate WES and RNAseq data obtained before therapy (V1T), after 12 weekly paclitaxel treatments (V2T) and after anthracycline-based regimen at surgical resection (V3T) to study a single patient with persistent TNBC disease after neoadjuvant chemotherapy. RESULTS Using RNA-Seq data, we identified an inter-chromosomal fusion transcript between chromosome 20 and 22 (GNAS-TTC38) that was highly expressed at V1T, V2T and V3T. We also identified intra-chromosomal fusion transcripts that were expressed at two time points, such as fusion transcript (KANSL1-ARL17A) on chromosome 17 for V1T and V2T and fusion transcript (RBM12B-LINC00535) on chromosome 8 for V2T and V3T time points. Several gene expression changes were also observed. Gene expression analysis of V1T, V2T and V3T tumors was performed. Differential temporal gene expression profiles of 9884 genes that were significant and varying at different time points were obtained for pathway analysis. Pathway analysis of 9884 genes identified up regulation and down regulation of several transcription factors with a fold change of 2x or more. When compared to blood, DNA tumor and RNA-Seq data, we identified 81 common somatic eSNVs that were expressed in both V1T and V2T time points and we are in the process of investigating V3T data. We found alterations of key transporter domains (CD225, Coatamer_beta_C, DUF2435, Dynamitin, EI24, GLTP, LMF1, Porin_3, V-ATPase_C) in our V1T and V2T SNV data. Similar to gene expression analysis, we are in the process of obtaining the list of mutations at various time points to identify driver and passenger mutation candidate genes for this specific TNBC patient. CONCLUSIONS Our initial time-series analysis of eSNV, fusion transcripts and gene expression data demonstrate that intensive analysis for individual patients is feasible. Further investigation of drug transporters and transcription regulators may help develop personalized treatment strategies for patients with disease resistant to current regimens. Citation Format: Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Douglas W. Mahoney, Poulami Barman, Jason P. Sinnwell, Hugues Sicotte, Peter Vedell, Steven N. Hart, Travis J. Dockter, Katie N. Jones, Amy L. Conners, Ann M. Moyer, Daniel W. Visscher, Jia Yu, Bowen Gao, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Jean-Pierre A. Kocher, Eric D. Wieben, Gianrico Farrugia, Cloann G. Schultz, James N. Ingle, Richard Weinshilboum, Matthew P. Goetz, Liewei Wang, Judy C. Boughey. Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2014-4185

Abstract Background Cancer genomic investigations have identified recurrent genomic aberrations critical for cancer initiation, progression, and metastases. However, these investigations are typically performed in isolation, and the effects of treatment on the clonal selection of tumor cells are mostly unknown. We hypothesized that molecular profiling of residual tumors after neoadjuvant chemotherapy (NAC) would identify new drug targets/pathways in patients at high risk for disease recurrence. To better identify clonal populations of resistant breast cancer cells, we utilized DNA content-based flow sorting of nuclei to identify and isolate clonal populations for aCGH and next generation sequencing (NGS) both before and after NAC. Methods The Breast Cancer Genome Guided Therapy Study (BEAUTY) (NCT 02022202) is a prospective study of patients with high-risk breast cancer treated with neoadjuvant 12 weekly paclitaxel (T) +/- trastuzumab followed by 4 cycles of anthracycline based chemotherapy. Tumor tissue from baseline, residual disease from surgery, distant metastases, and patient derived xenografts (PDX) are obtained for cell sorting by DNA ploidy, aCGH, RNA and exome sequencing. Results: 140 patients have been enrolled, 104 have completed surgery and 30 unique PDX have been established corresponding to 26 patients prior to chemotherapy and 4 from residual disease at surgery. Baseline exome and RNA sequencing is complete in 140. Currently, genomic analyses of flow sorted matched baseline, surgical, PDX, and distant disease samples are available in 6 patients. Substantial genomic variation was observed in the surgical sample compared to the primary tumor including gain of oncogenic drivers (EGFR) and loss of negative regulators (ATG5) (Table). The PDX recapitulated these events with excellent fidelity compared to the corresponding human tumor. In patients with TNBC, RNA seq obained from matched samples demonstrated changes in immune related pathways. Evaluation of drug targets/pathways identified in the resistant tumors are ongoing using the PDX and sequencing of the remaining matched baseline/surgical disease will be reported. Clonal changes occurring over time in patients with residual disease or disease recurrence after NACTumor SubtypeResidual Cancer BurdenDisease StatusClonal Aberration Changes (baseline and post NAC)TNBC (AR subtype)3Contralateral lymph node recurrence at 150 days2p25.2 - p25.1 amplicon lost at recurrence; 6p21.32 -p21.31 amplicon lost at recurrenceTNBC (BL 1)3Bone/liver/lymph node recurrence at 135 days5q11.2 amplicon gained at surgery; 12p13.33 - p13.2 amplicon gained at surgeryTNBC (BL 1)3Disease-free at day 1506q21 amplicon lost at sugeryTNBC (BL 2)0Brain recurrence at 390 daysChr 2 chromothripsis at surgeryLuminal B3Progression during chemotherapy7p12.1 - p11.2 amplicon gained at surgeryLuminal HER23Disease-free at day 3609q33.2 amplicon lost at surgery; 15q13.1 - q13.3 amplicon lost at surgery; 15q22.2 - q24.1 amplicon lost at surgery Conclusions: We observed substantial evolutionary changes in residual breast tumors remaining after NAC. Our findings suggest that a comprehensive assessment of the mutational landscape that has evolved during NAC can inform drug development in high risk breast cancer patients. Citation Format: Matthew P Goetz, Michael T Barrett, Krishna R Kalari, Vera J Suman, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Ann M Moyer, Donald W Northfelt, Richard J Gray, Jason Sinnwell, Douglas Mahoney, Poulami Barman, Peter Vedell, Xiaojia Tang, Kevin Thompson, Travis Dockter, Katie Jones, Sara J Felten, Amy Conners, Jeanette Eckel-Passow, Hughes Sicotte, Steven N Hart, Jia Yu, Daniel W Visscher, Eric D Wieben, Cloann Schultz, Minetta C Liu, James N Ingle, Liewei Wang, Richard W Weinshilboum, Judy C Boughey. Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer [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 PD3-3.

Abstract Objective: Contralateral PM (CPM) and Bilateral PM (BPM) markedly decrease, but do not completely eliminate the possibility of development of a new BC on the side of the PM. Given the relative infrequency of its occurrence, little is known about the clinical characteristics, presentation, and management of patients who develop BC after PM. Our aim was to review our institutional experience of BC occurring after PM. Methods: Between 1960 and 1993, 1,065 women underwent BPM and 1,643 women with unilateral BC treated with therapeutic mastectomy underwent a CPM. Medical records were reviewed and study-specific questionnaires were sent to all women at 10 years and 20 years after PM. BC after PM included locoregional invasive BC or DCIS on the side of the PM. Results: Thirteen patients who underwent BPM developed BC after PM. Twelve patients who underwent CPM developed a subsequent BC on the side of the CPM. The median follow-up time from PM was 22 years (range 3-34). Detailed clinical characteristics of BC after PM are shown in Table 1. Presentations included: disease limited to the axilla without evidence of a local primary 4 (16%); synchronous local and axillary disease 1 (4%); synchronous local disease and distant metastases 4 (16%); clinically isolated local disease 17 (68%). Characteristics of BC Following PM All Patients (n=25)BPM Cohort (n=13)CPM Cohort (n=12)Median age at diagnosis of BC after PM56 (range 38-81)58 (range 38-71)54 (range 39-81)Median time to development of BC after PM (years)7 (range 1-25)6 (range 2-25)8 (range 1-21)Presentation Self-detected abnormality23 (92%)12 (92%)11 (92%)Screening mammogram1 (4%)02 (16%)Not known1 (4%)1 (8%)0Local disease only17 (68%)10 (77%)7 (58%)-Sub-areolar7 (41%)6 (40%)1 (8%)-UOQ/axillary tail2 (12%)02 (16%)-Lower/inframmamary crease2 (12%)1 (10%)1(8%)Chest wall or unspecified6 (35%)3 (30%)3 (25%)Local & regional (axillary) disease1 (4%01 (8%)Axillary BC without evidence of local primary4 (16%)1 (8%)3 (25%)Synchronous local and distant disease3 (12%)2 (15%)1 (8%) Of the 17 patients with isolated local disease, 11 (65%) underwent a completion/redo mastectomy, local excision of the tumor was performed in 5 (29%), and surgical management was unknown in 1 (6%). Ten of 17 (59%) underwent axillary lymph node dissection, 1 (6%) underwent sentinel lymph node biopsy, 1 did not undergo axillary staging, and axillary management was unknown in 5 (29%). Median tumor size was 0.9 cm (range 0.3-3.5) and only 1 of 17 (6%) patients was confirmed to have pathologic nodal involvement. Twelve of 17 (71%) received some type of adjuvant therapy: chemotherapy and/or endocrine therapy 3 (18%); radiotherapy 2 (12%); both 5 (29%); none 5 (29%). With a median follow-up of 7 years since diagnosis of local BC after PM, there has been one isolated local recurrence and 2 distant recurrences as first event. Conclusion: BC can occur after PM. With rising rates of PM, understanding management of BC after PM is important. Most common presentation is local disease and can be managed with resection with consideration of adjuvant therapy. Multidisciplinary management of these cases is needed. Citation Format: Robert W Mutter, Tanya L Hoskin, Marlene H Frost, Joanne L Johnson, Lynn C Hartmann, Judy C Boughey. Breast cancer (BC) following prophylactic mastectomy (PM), a clinical entity: Presentation, management, and outcomes [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 P5-12-05.

Abstract Background: Although breast cancer (BC) may occur at any age, its prevalence is greater postmenopause. Greater than 75 % of postmenopausal BCs are hormone dependent. Aromatase inhibitors suppress postmenopausal estrogen biosynthesis. Letrozole has demonstrated efficacy against BC in the adjuvant and metastatic setting at the treatment dose of 2.5 mg daily. Its potential role in BC prevention has been inferred from reductions in contralateral BCs. Its side effect profile, similar to other AIs, includes exacerbation of menopausal symptoms that negatively impact quality of life (QOL) and may result in discontinuation of the drug. Both anastrazole and exemestane have been demonstrated to reduce BC in high-risk women. Hypothesis: Lower and intermittent doses of letrozole effectively suppress estrogen in the high-risk postmenopausal woman and provide a better side effect profile. Methods: A randomized, double-blind study comparing the impact of varying letrozole doses (2.5 mg daily, 2.5 mg MWF, 1.0 mg MWF, or 0.25 mg MWF) on estrogen suppression and side effects--lipids, bone resorption, menopause, and QOL-- was conducted. Participants randomized to intermittent dosing received placebo on nontreatment days. Results: 112 participants were enrolled at 2 clinical sites. Mean patient age was 62.8 years, and average BMI was 29.8. Analysis of available data after 24 weeks of therapy revealed statistically significant increase in triglycerides (N=94): 114.67±48.39 to 125.79±54.31 (p<0.01); vasomotor symptoms (N=95): 2.25±1.26 to 2.74±1.67 (p<0.01); and C-telopeptide (N=75): 0.39±0.23 to 0.55±0.28 (p<0.0001). Statistically significant decrease in estradiol (N=68): 5.57±5.19 to 1.26±1.41 (p<0.0001) and estrone (N=68): 22.39±13.02 to 1.64±2.66 (p<0.0001) were observed. No differences in QOL (SF 36) were noted after letrozole treatment. P-values were derived from paired t-test (signed rank test) for the difference between baseline and after 24 weeks of study drug. Conclusions: De-escalating doses of letrozole suppress postmenopausal estrogen effectively and result in statistically significant increases in triglycerides, C-telopeptide and vasomotor symptoms without impact on QOL. Presentation will include unblinded intervention arm outcomes. Citation Format: Ana Maria Lopez, Hsiao Hui Sherry Chow, Denise Frank, Sandhya Puthi, Judy Boughey, Paul Hsu, Jose Guillen, Marjorie Perloff, Michelle Ley, Julie E Lang. De-escalating doses of letrozole in post menopausal women at high risk for breast cancer [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 P5-12-04.

Abstract Background: Further stratification of the 70-gene MammaPrintTM signature into ‘high’ and ‘ultra-high’ risk groups may help predict chemo-sensitivity. In I-SPY 2, patients were classified as MammaPrint High1 (MP1) or MammaPrint (ultra) High2 (MP2), with MP2 defined as MP_score <-0.154. MP1/MP2 classification was added to HR and HER2 to define the cancer subtypes used in the I-SPY 2 adaptive randomization engine. Neratinib (N), one of the experimental agents evaluated in I-SPY 2, graduated in the HR-HER2+ signature. All patients received at least standard chemotherapy (paclitaxel followed by doxorubicin/cyclophosphamide; T->AC). HER2- patients were randomized to receive N+T- >AC vs. T->AC. For HER2+ patients, neratinib was administered in place of trastuzumab (N+T->AC vs. H+T->AC). Here, we assess the performance of MP1/MP2 class as a specific biomarker of neratinib response. Methods: 115 patients in the neratinib arm and 76 concurrently randomized controls had Agilent 44K microarrays and pCR data available for analysis. We assess association between MP1/MP2 and response in the neratinib and control arms alone using Fisher’s exact test, and relative performance between arms (biomarker x treatment interaction, likelihood ratio p < 0.05) using a logistic model. This analysis is also performed adjusting for HR status as a covariate, and in receptor subsets. Our study is exploratory with no claims for generalizability of the data. Statistical calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Our analyses do not adjust for multiplicities of other biomarkers in the trial but outside this study. Results: There are 133 MP1 patients (neratinib: 74, Control: 59) and 58 MP2 patients (neratinib: 41, Control: 17), 84% (49) of which are Her2-. The distribution of pCR rates among MP1/MP2 dichotomized groups are summarized in Table 1. Neratinib (n=115)Control (n=76)MP1 (n=74)MP2 (n=41)MP1 (n=59)MP2 (n=17)HER2- (n=105)0 / 1715 / 337 / 395 / 16HER2+ (n=86)22 / 574 / 85 / 200 / 1 MP2, one of the 10 eligible signatures, did not meet the graduation threshold; and MP1/MP2 did not show a significant biomarker x treatment interaction (OR in neratinib relative to control arm = 1.25). The MP1/MP2 x treatment interaction remains non-significant after adjustment for HR and HER2 status (p=0.54). In HER2- patients receiving neratinib, 45% (15/33) of MP2 patients achieved a pCR, compared to 0% (0/17) of MP1 patients. In the HER2- controls, there is a 31% pCR rate in MP2 (5/16) vs. 18% in MP1 (7/39) patients (OR=2.14). This difference in performance between treatment arms appears significant (p=0.041). 90% of HER2+ patients are MP1, thus MP1/MP2 status x treatment interaction within the HER2+ subtype cannot be evaluated. Conclusion: Within the I-SPY 2 population as a whole, MP1/MP2 stratification does not appear to be a specific biomarker of response to neratinib relative to the control arm. The number of HER2- patients is small and precludes any definitive conclusion, but these data motivate further investigation of the biological mechanisms distinguishing MP1 from MP2 to better understand chemotherapy and/or neratanib responsiveness. Citation Format: Christina Yau, Denise M Wolf, Ashish Sanil, Jo Chien, Anne Wallace, Judy Boughey, Doug Yee, Debu Tripathy, Angela DeMichele, Rita Nanda, Steven Chiu, Claudine Isaacs, Kathy Albain, Hank Kaplan, Stacey Moulder, Rebecca Viscusi, Donald Northfelt, Kirsten Edmiston, Anthony Elias, Toncred Styblo, Barbara Haley, Lamorna Brown-Swigart, Susan Flynn, Gillian L Hirst, Meredith Buxton, Nola Hylton, Melissa Paoloni, W Fraser Symmans, Laura Esserman, Don Berry, Minetta C Liu, John W Park, Laura van 't Veer. MammaPrint High1/High2 risk class as a biomarker of response to neratinib plus standard neoadjuvant therapy for breast cancer in the I-SPY 2 TRIAL [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 P3-06-29.

Abstract Background: Further stratification of the 70-gene MammaPrintTM signature into ‘high’ and ‘ultra-high’ risk groups may help predict chemo-sensitivity. In I-SPY 2, patients were classified as MammaPrint High1 (MP1) or MammaPrint (ultra) High2 (MP2), with MP2 defined as MP_score <-0.154. MP1/MP2 classification was added to HR and Her2 to define the cancer subtypes used in the I-SPY 2 adaptive randomization engine. HER2- patients were randomized to receive standard chemotherapy or the oral PARP inhibitor veliparib in combination with carboplatin (V/C) and chemotherapy. V/C graduated in the triple-negative (TN) signature, where MP2 was not an eligible signature for graduation. Here, we assess the performance of MP1/MP2 class as a specific biomarker of response to V/C. Methods:115 HER2- patients (V/C: 71 and concurrent controls: 44) were considered in this analysis. We assess association between MP1/MP2 and response in the V/C and control arms alone using Fisher’s exact test, and relative performance between arms (biomarker x treatment interaction, likelihood ratio p < 0.05) using a logistic model. This analysis is also performed adjusting for HR status as a covariate. To assess MP1/MP2 in the context of the graduating signature, we added the MP2 patients to the graduating TN subset and evaluated the treatment effect in this ‘biomarker-positive’ group. Our study is exploratory with no claims for generalizability of the data. Statistical calculations are descriptive (e.g. p-values are measures of distance with no inferential content). This analysis does not adjust for multiplicities of other biomarkers in the trial but outside this study. Results: In the V/C arm vs. concurrent controls, there were 66 MP1 (V/C: 32, Control: 34) and 49 MP2 patients (V/C: 39, Control: 10), 78% of which are TN. The distribution of pCR rates among MP1/MP2 dichotomized groups are summarized in Table 1. V/C (n=71)Control (n=44)MP1 (n=32)MP2 (n=39)MP1 (n=34)MP2 (n=10)TN (n=59)3 / 819 / 303 / 132 / 8HR+HER2- (n=56)1 / 244 / 94 / 210 / 2 The OR between MP1/MP2 risk groups for predicting pCR is 9.71 in the V/C arm (p=6.63E-05), in comparison to an OR of 0.97 in the control arm (p=1). There is a significant biomarker x treatment interaction (p=0.023), which remains upon adjusting for HR status (p= 0.028). Based on the I-SPY 2 Bayesian model, a Phase III trial with 300 MP2 patients has a 95% predictive probability of success. When the MP2 patients are added to the graduating TN subset, the OR associated with V/C is 4.36, which is comparable to that of the TN signature (OR: 4.29), while increasing the prevalence of biomarker-positive patients by ∼10%. Conclusion: In our exploratory analysis, MP2 suggests higher sensitivity to V/C combination therapy relative to controls. This observation has prompted an investigation into the biological mechanisms distinguishing the MP1/MP2 subtype that may account for this specificity. Citation Format: Denise M Wolf, Christina Yau, Ashish Sanil, Jo Chien, Anne Wallace, Angela DeMichele, Hank Kaplan, Doug Yee, Claudine Isaacs, Kathy Albain, Rebecca Viscuzi, Judy Boughey, Stacey Moulder, Steven Chui, Qamar Khan, Toncred Styblo, Kirsten Edmiston, Donald Northfelt, Anthony Elias, Barbara Haley, Debu Tripathy, Lamorna Brown-Swigart, Susan Flynn, Gillian Hirst, Meredith Buxton, Nola Hylton, Melissa Paoloni, Fraser Symmans, Laura Esserman, Don Berry, Hope Rugo, Olufunmilayo I. Olopade, Laura van 't Veer. MammaPrint High1/High2 risk class as a biomarker of response to veliparib/carboplatin plus standard neoadjuvant therapy for breast cancer in the I-SPY 2 TRIAL [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 P3-06-25.

Abstract Background: The American College of Surgeons Oncology Group (ACOSOG) Z1071 trial (Alliance) reported a false negative rate (FNR) of 12.6% with sentinel lymph node (SLN) surgery after neoadjuvant chemotherapy in women presenting with node-positive breast cancer. Proposed methods to decrease the FNR include clip placement in the positive node at initial diagnosis with confirmation of resection of the clipped node at surgery and inclusion of residual metastatic cells identified by immunohistochemistry (IHC) for cytokeratins and disease measuring <0.2mm on H&E in the definition of node positivity after chemotherapy. Herein we evaluate the impact of these methods on FNR. Methods: Z1071 was a prospective multi-institutional trial in which women with clinical T0-4,N1-2,M0 breast cancer underwent both SLN surgery and axillary dissection (ALND) after neoadjuvant chemotherapy. The primary endpoint defined nodal metastasis as disease >0.2mm on H&E. Slides were submitted for central IHC staining when H&E-negative, unless performed locally. For this analysis, we expanded the definition of node positivity to include metastases less than 0.2mm on H&E and metastatic deposits identified by IHC. In 171 cases, a clip was placed in the node at initial biopsy and in these cases the SLNs and ALND were evaluated by x-ray or pathology to document clip location. FNR in the clip cohort is reported using the primary endpoint definition of node positive disease (>0.2mm on H&E). Results: In the 113 cases where the clip was found in the SLN the FNR was 6.4% (1.8-15.5%). In the 29 cases where the clip was found in the ALND specimen, the FNR was 22.2% (6.4-47.6%). IHC was available on 470 of 525 patients with cN1 disease and 2 SLNs resected. Using the definition of H&E metastasis >0.2mm, the FNR in these patients was 11.3% (34/301, 8.0-15.4%) which decreased to 8.7% (27/311, 5.6-11.8%) when including any disease <0.2mm. SLNs from 16 patients had disease <0.2mm in size. Seven patients previously classified as false negative SLN changed to true positive with identification of disease in the SLN. Nine patients changed from node-negative to node-positive with the only disease being <0.2mm disease found in the SLN. Nodal pathologic complete response rate changed from 36.0% to 33.8% with the inclusion of metastases <0.2mm. NResidual disease identified in SLNs or ALNDFNR (%)95% CINode positive definitionSLN metastases >0.2mm by H&E470301 (64.0%)11.38.0-15.4SLN metastasis any size on IHC or H&E470311 (66.2%)8.75.6-11.8Clip locationClip in SLN11462 (54.4%)6.41.8-15.5Clip in ALND2918 (62.1%)22.26.4-47.6Clip location unknown2920 (69.0%)14.33.0-36.3Clip not placed354207 (58.5%)13.59.1-18.9 Conclusion: Placement of a clip at initial diagnosis of node positive disease with identification of the clip during the SLN surgery reduces the FNR. Use of IHC with inclusion of metastases <0.2mm in the definition of residual nodal disease in women with node positive breast cancer after chemotherapy also improves the accuracy of SLN surgery. Use of one or both of these methods should be considered when performing SLN in this setting. Citation Format: Judy C Boughey, Karla V Ballman, William F Symmans, Linda M McCall, Elizabeth A Mittendorf, Gretchen M Ahrendt, Lee G Wilke, Bret Taback, Kelly K Hunt. Methods impacting the false negative rate of sentinel lymph node surgery in patients presenting with node positive breast cancer (T0-T4,N1-2) who receive neoadjuvant chemotherapy – Results from a prospective trial – ACOSOG Z1071 (Alliance) [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 P2-01-02.

Abstract Background: Many newly diagnosed breast cancer patients undergo preoperative breast MRI which includes visualization of some degree of the axilla. The impact of patient characteristics and tumor biology on the fidelity of MRI imaging of the axilla in these cases is not well-studied. We sought to examine the correlation between nodal findings on breast MRI and pathologic nodal status in newly diagnosed breast cancer patients. Methods: With IRB approval, we identified 1868 consecutive breasts with invasive cancer in 1803 patients undergoing primary operation with axillary surgery from 1/2010-7/2013. Patients undergoing neoadjuvant chemotherapy (348) were excluded leaving 1510 breasts with cancer in 1455 patients. Preoperative MRI was performed in 763 patients (52%). We evaluated patient, imaging and pathology data. Results: Patients evaluated with MRI were median age 57 years. The majority of tumors were T1 (63%) and T2 (28%). MRI identified suspicious axillary nodes in 240 cases (31%), of which 123 (51%) were node positive at operation. Suspicious axillary findings on MRI predicted both nodal status and pN stage at operation (both p<0.0001). The sensitivity, specificity, accuracy, positive (PPV) and negative predictive value (NPV) of MRI for nodal disease across tumor types is shown. Pathologic Node PositiveSensitivity (95% CI)Specificity (95% CI)NPV (95% CI)PPV (95% CI)Accuracy (95% CI)All Cases (n=763)234 (31%)52.6 (46-59.1)77.9 (74.1-81.3)78.8 (75-82.1)51.3 (44.8-57.7)70.1 (66.7-73.3)Approximated Biologic Subtype (missing=24)ER+/HER2- (n=609)194 (32%)51.0 (43.8-58.2)80.0 (75.8-83.7)77.8 (73.4-81.5)54.4 (46.9-61.7)70.8 (67-74.3)HER2+ (n=69)20 (29%)65.0 (40.9-83.7)71.4 (56.5-83)83.3 (68-92.5)48.1 (29.2-67.6)69.6 (57.2-79.8)ER-/HER2- (n=61)17 (28%)58.8 (33.5-80.6)75.0 (59.4-86.3)82.5 (66.6-92.1)47.6 (26.4-70)70.5 (57.3-81.1)p value0.740.430.330.560.730.98Tumor Grade (missing=5)1 (n=259)58 (22%)32.8 (21.4-46.5)79.6 (73.2-84.8)80.4 (74.1-85.5)31.7 (20.6-45.1)69.1 (63.0-74.6)2 (n=351)124 (35%)55.6 (46.5-64.5)79.7 (73.8-84.6)76.7 (70.7-81.8)60.0 (50.4-68.9)71.2 (66.1-75.9)3 (n=148)51 (34%)66.7 (52.0-79.0)70.1 (59.8-78.8)80.0 (69.6-87.6)54.0 (41.0-66.4)68.9 (60.7-76.1)p value0.0010.00030.110.720.020.93 Diagnostic performance did not vary significantly based on patient body mass index (BMI) or approximated biologic subtype, but specificity was better for patients >age 50 (p=0.007) and sensitivity and PPV both were worse for grade 1 vs grade 2/3 tumors. Node positivity rate at operation was 48% (51/106) for patients with a solitary abnormal node on MRI and 54% (72/134) when >1 suspicious node was seen (p=0.39). However, multiple vs solitary MRI suspicious nodes correlated with ≥3 positive nodes at operation (40 [30%] vs 19 [18%], p=0.03) and pN2/pN3 disease (29 [22%] vs 12 [11%], p=0.03). Conclusion: Axillary lymph node findings on MRI for breast cancer predict nodal status and disease volume in invasive breast cancer patients. Tumor biologic subtype did not affect performance characteristics of preoperative MRI, but tumor grade did influence the sensitivity and PPV of MRI. When MRI is performed in the evaluation of newly diagnosed breast cancer, axillary findings inform pathologic nodal stage at operation regardless of tumor subtype. Citation Format: Sean C Dupont, Judy C Boughey, Tanya L Hoskin, Katrina N Glazebrook, Tina J Hieken. Value of breast MRI for preoperative axillary assessment of breast cancer patients [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 P1-01-03.

Abstract Background: I-SPY 2 is a multicenter, phase II neoadjuvant trial in women with high-risk stage II/III breast cancer using adaptive randomization within biomarker subtypes to evaluate novel agents added to standard chemotherapy. Primary endpoint is pathologic complete response (pCR). Goal is to identify regimens that meet a high Bayesian predictive probability of statistical significance in a neoadjuvant 300-patient phase III trial defined by hormone-receptor (HR), HER2 status, and MammaPrint (MP). Experimental regimens may “graduate” in 1 of 10 signatures, with a maximum of 120 patients. We report efficacy results for neratinib (N). Methods: Tumors ≥2.5cm by clinical exam & ≥2cm by imaging are eligible for screening. MP low risk/HR+/HER2- tumors are ineligible for randomization. Patients receive chemotherapy (paclitaxel qwk x 12, doxorubicin and cyclophosphamide q2-3 wk x 4, T->AC). HER2- pts were randomized to N+T->AC vs. T->AC and HER2+ pts to N+T->AC vs. trastuzumab+T->AC. Analysis is intent-to-treat with pts who switch to non-protocol therapy regarded as non-pCRs. We provide estimated pCR rates (95% Bayesian probability intervals), probabilities of superiority of neratinib over control, and Bayesian predictive probabilities of success in an equally randomized phase III trial. Results: Neratinib met the predictive probability criterion in HR-/HER2+, “graduated”, and accrual ceased [115 N patients (65 HER2+), 78 concurrently randomized controls (22 HER2+)]. The table shows results for all 10 signatures. Two patients (1 N and 1 control) withdrew consent and are not included. Conclusion: I-SPY 2's standing trial mechanism efficiently evaluates agents in biomarker-defined patient subsets. In a modest number of patients, adaptive randomization successfully identified a biomarker signature (HR-/HER2+) for neratinib's further development. All HER2+ and MP+ tumors may also benefit from this regimen, consistent with preclinical data. Evaluation in I-SPY 3, a phase III registration trial, is planned. SignatureEstimated pCR Rate (95% probability interval)Probability NeratinibIs Superior to ControlPredictive Probability of Success in Phase IIINeratinibControlALL32% (28-36%)23% (19%-28%)92%44%HR+23% (18%-28%)17% (11%-22%)81%40%HR-43% (37%-49%)31% (24%-38%)89%53%HER2+39% (33%-45%)23% (15%-30%)95%73%HER2-26% (21%-32%)24% (18%-29%)63%20%MP+*45% (38%-53%)29% (20%-39%)91%66%HR-/HER2-36% (29%-43%)30% (23%-38%)72%34%HR-/HER2+55% (46%-64%)32% (22%-43%)94%78%HR+/HER2+31% (24%-37%)17% (10%-24%)91%65%HR+/HER2-14% (8%-19%)16% (10%-21%)39%12%* MammaPrint 44K Array Low/High score was adjusted to MammaPrint High1 (MP-) or High2 (MP+), using a pre-defined median cut-point of I-SPY 1 participants, who fit the eligibility criteria of I-SPY 2. Citation Format: John W. Park, Minetta C. Liu, Douglas Yee, Angela DeMichele, Laura van 't Veer, Nola Hylton, Fraser Symmans, Meredith B. Buxton, A. Jo Chien, Amy Wallace, Michelle Melisko, Richard Schwab, Judy Boughey, Debashish Tripathy, Hank Kaplan, Rita Nanda, Stephen Chui, Kathy S. Albain, Stacy Moulder, Anthony Elias, Julie E. Lang, Kirsten Edminston, Donald Northfelt, David Euhus, Qamar Khan, Julia Lyandres, Sarah E. Davis, Christina Yau, Ashish Sanil, Laura J. Esserman, Donald A. Berry. Neratinib plus standard neoadjuvant therapy for high-risk breast cancer: Efficacy results from the I-SPY 2 TRIAL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT227. doi:10.1158/1538-7445.AM2014-CT227

Abstract INTRODUCTION Patient derived xenografts (PDX) may better reflect individual patient (pt) tumor biology; however, the feasibility of collecting PDX from percutaneous tumor biopsies (PTB) in the neoadjuvant setting is unknown. Furthermore, drug response phenotypes observed in PDX have not been prospectively compared to the corresponding pt clinical outcomes. METHODS The Breast Cancer Genome Guided Therapy Study (BEAUTY) is a prospective Mayo study of pts with high-risk breast cancer treated with neoadjuvant weekly paclitaxel (T) +/- trastuzumab followed by anthracycline based chemotherapy. PTB (at baseline) and residual surgical tissue (after all chemotherapy) are obtained for next generation sequencing (NGS) and PDX. Tumor biopsies (1-2 cores from 14 gauge needle) were implanted with Matrigel <1 hour of collection in the flanks of NOD-SCID or NSG mice. Low dose estradiol was supplemented in the drinking water. Primary outgrowth rate was defined as PDX tumor volume >50 mm3. Take rate was defined as development of at least 1 stably transplantable xenograft line/pt. To determine whether clinical T response assessed by MRI corresponded with in vivo T response, pretreatment PDX from 5 pts were injected into NOD-SCID mice (20 mice per pt PDX) and when tumors reached 100-200mm3, mice were randomized to no treatment vs T (20 mg/kg, ip. every 3-4 days). Two of these 5 patients had a MRI response defined as >30% decrease in longest lesion. RESULTS Pretreatment PTB from 81 unique pts were implanted in 251 mice (2-4 mice/pt). PDX outgrowth rates were 33.3% (27/81 pts) and 22 stable PDX were established (overall take rate 27.2%). Take rates were as follows: triple negative breast cancer (46%; 13/28); HER2 (27%; 6/22), Luminal B (13%; 3/22), and luminal A (0%; 0/9). Residual surgical tumor (after all treatment) from 17 pts was injected into 85 mice (average 5 mice/pt) and the initial outgrowth rate was 23% (4/17) with 3 stably transplantable lines established. PDX, derived from pretreatment PTB of 5 pts (2 responders and 3 non-responders), were assessed for in vivo T response. The size of the T treated group was significantly smaller than the no treatment group for the PDX derived from the 2 clinical responders, with complete disappearance of tumor by 18 days. In contrast, the PDX derived from the 3 clinical non-responders had no evidence for T response. CONCLUSIONS We have demonstrated the feasibility of using PTB to establish PDX in a prospective neoadjuvant clinical study and have demonstrated similar T drug response phenotypes in in the PDX as seen in the corresponding pt. These data suggest that PDX generated prospectively may be useful for biomarker validation and the development and individualization of new drug therapy. Funded by the Mayo Clinic Center for Individualized Medicine and the Mayo Clinic Cancer Center Citation Format: Jia Yu, Ping Yin, Bowen Gao, Jason P. Sinnwell, Ann M. Moyer, Daniel W. Visscher, Amy L. Conners, Travis J. Dockter, Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Hugues Sicotte, Douglas W. Mahoney, Steven N. Hart, Peter T. Vedell, Poulami Barman, Katie N. Jones, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Eric D. Wieben, James N. Ingle, Zhenkun Lou, Gianrico Farrugia, Richard Weinshilboum, Matthew P. Goetz, Judy C. Boughey, Liewei Wang. Feasibility of using percutaneous tumor biopsies from a prospective neoadjuvant breast cancer study to develop patient derived xenografts and assess in vivo chemotherapy sensitivity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2014-1195