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Tumors are increasingly appreciated as complex ecosystems, wherein functional interactions between tumor subclones, as well as components of the microenvironment, contribute to progression and drug resistance. While some studies have focused on soluble factors that mediate these interactions, little is known about the communication between physically neighboring tumor subclones (and their microenvironment). To investigate the nature and impact of such localized interactions, we are assessing the activation status of key cancer signaling pathways in neighboring tumor cells; importantly, we are characterizing these features at the single cell level within physically intact tumors. Triple negative breast cancer (TNBC) exhibits a high degree of intratumor heterogeneity, which has contributed to a lack of effective targeted therapy options. Chemotherapy remains the standard of care; however approximately half of patients have substantial residual disease following chemotherapy, which is associated with a high risk of recurrence. Our objective is to spatially define signaling heterogeneity using patient-derived xenograft (PDX) models before and after chemotherapy treatment, to determine if spatially-defined signaling niches drive chemoresistance in TNBC. We hypothesize that neighborhoods of cells possess specialized phenotypes that mediate chemoresistance, and when disrupted, will inhibit the growth of chemoresistant tumors. We are employing imaging mass cytometry (IMC),a next-generation immunostaining approach that allows for simultaneous measurement of 30-40 biomarkers while retaining the spatial organization of the sample.Wehave constructed an IMC panel of antibodies that combines markers for tissue architecture, tumor and immune cell phenotyping, and signaling pathway activation. Our PDX collection features sequential pairs derived from biopsies taken before and after chemotherapy treatment. IMC of the pre-/post-chemotherapy pairs revealed spatial patterns of pathway activation that emerged following treatment, including increases in PI3K/mTOR and localized MAPK signaling. To complement these studies, we treated PDX models with chemotherapy and analyzed tumors via IMC throughout the course of treatment. Again, we observed the emergence of increased MAPK signaling, localized to discrete neighborhoods within the tumor. Taken together, our findings suggest that unique signaling niches arise following treatment. Our goal is to now determine whether these signaling niches functionally contribute to chemoresistance and if disruption of these niches can inhibit the growth of chemoresistant disease. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Gloria V. Echeverria, Xiaomei Zhang, Jiansu Shao, W. Fraser Symmans, Stacy L. Moulder, Helen Piwnica-Worms. Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1513.

Importance Approximately 25% of patients with early-stage breast cancer who receive (neo)adjuvant chemotherapy experience a recurrence within 5 years. Improvements in therapy are greatly needed. Objective To determine if pembrolizumab plus neoadjuvant chemotherapy (NACT) in early-stage breast cancer is likely to be successful in a 300-patient, confirmatory randomized phase 3 neoadjuvant clinical trial. Design, Setting, and Participants The I-SPY2 study is an ongoing open-label, multicenter, adaptively randomized phase 2 platform trial for high-risk, stage II/III breast cancer, evaluating multiple investigational arms in parallel. Standard NACT serves as the common control arm; investigational agent(s) are added to this backbone. Patients withERBB2(formerlyHER2)-negative breast cancer were eligible for randomization to pembrolizumab between November 2015 and November 2016. Interventions Participants were randomized to receive taxane- and anthracycline-based NACT with or without pembrolizumab, followed by definitive surgery. Main Outcomes and Measures The primary end point was pathologic complete response (pCR). Secondary end points were residual cancer burden (RCB) and 3-year event-free and distant recurrence-free survival. Investigational arms graduated when demonstrating an 85% predictive probability of success in a hypothetical confirmatory phase 3 trial. Results Of the 250 women included in the final analysis, 181 were randomized to the standard NACT control group (median [range] age, 47 [24.77] years). Sixty-nine women (median [range] age, 50 [27-71] years) were randomized to 4 cycles of pembrolizumab in combination with weekly paclitaxel followed by AC; 40 hormone receptor (HR)-positive and 29 triple-negative. Pembrolizumab graduated in all 3 biomarker signatures studied. Final estimated pCR rates, evaluated in March 2017, were 44% vs 17%, 30% vs 13%, and 60% vs 22% for pembrolizumab vs control in theERBB2-negative, HR-positive/ERBB2-negative, and triple-negative cohorts, respectively. Pembrolizumab shifted the RCB distribution to a lower disease burden for each cohort evaluated. Adverse events included immune-related endocrinopathies, notably thyroid abnormalities (13.0%) and adrenal insufficiency (8.7%). Achieving a pCR appeared predictive of long-term outcome, where patients with pCR following pembrolizumab plus chemotherapy had high event-free survival rates (93% at 3 years with 2.8 years’ median follow-up). Conclusions and Relevance When added to standard neoadjuvant chemotherapy, pembrolizumab more than doubled the estimated pCR rates for both HR-positive/ERBB2-negative and triple-negative breast cancer, indicating that checkpoint blockade in women with early-stage, high-risk,ERBB2-negative breast cancer is highly likely to succeed in a phase 3 trial. Pembrolizumab was the first of 10 agents to graduate in the HR-positive/ERBB2-negative signature. Trial Registration ClinicalTrials.gov Identifier:NCT01042379

Background: Recent studies have demonstrated independent validation of the prognostic relevance of residual cancer burden (RCB) after neoadjuvant chemotherapy. However, a pooled subject-level analysis of multiple cohorts is needed to determine estimates of long-term prognosis for each class of RCB in each phenotypic subtype of breast cancer (BC) to better inform on patient outcomes. Also, a pooled subject-level analysis allows more detailed analyses of generalizability of the prognostic meaning of RCB assessments in a broader experience of practice settings. Method: Subject-level RCB results, with relevant clinical and pathologic stage, tumor subtype and grade, demographic, treatment and follow-up data from 11 institutes/trials are being collected for combined analysis. The association between the continuous RCB index and event-free survival (EFS), and distant recurrence free survival (DRFS) were assessed using mixed effect Cox models with the incorporation of random RCB coefficients to account for between-study heterogeneity. We will also allow for differences in baseline hazard across biological BC subtypes and, if needed, across studies as well. In addition to this stratified mixed effect model, a multivariate analysis adjusting for age, T-category, nodal status and grade was performed within each subtype. In addition, mixed effect Cox models will be employed to evaluate association between RCB index with EFS and DRFS within each HR/HER2 subtype. Kaplan Meier estimates of EFS and DRFS at 5 and 10 years were computed for each RCB class within subtype. Results: We analyzed subject-level data from 9 institutes/trials representing 4077 patients currently available from an anticipated final total of 4,800 patients (to be presented at the meeting). There were 950 EFS and 876 DRFS events during follow up (median 65 months, IQR: 70 months). RCB index (continuous) was independently prognostic within each subtype: HR+/HER2- (EFS HR (per unit increase in RCB index) =1.64, 95%CI 1.48-1.82; DRFS HR=1.68, 1.51-1.87), HR+/HER2+ (EFS HR=1.80, 1.57-2.05; DRFS HR=1.93, 1.67-2.24), HR-/HER2+ (EFS HR=2.15, 1.76-2.62; DRFS HR=2.10, 1.77-2.50), and HR-/HER2- (EFS HR=2.05, 1.89-2.22; DRFS HR=2.16, 1.90-2.46); and remained prognostic in multivariate models adjusting for age, grade, and clinical T and N stage at diagnosis. Table 1 contains the response rate and estimated EFS at 5 years and 10 years for each RCB class within each HR/HER2 phenotype (DRFS results were similar). Conclusions: Long-term prognosis after pCR was similarly excellent in all phenotypic subtypes. RCB index and classification was independently and strongly prognostic in all subtypes, and generalizable to multiple practice settings. Prognostic differences by RCB class occurred within 5 years in HR- BC, but extended to 10 years in HR+ BC. RCB-I had slightly worse EFS than pCR in HR- BC and HR+/HER2+ BC (after 5 years), but the same EFS as pCR in HR+/HER2- BC. Complete analysis of all subjects, including neoadjuvant treatments, will be presented at the meeting. PhenotypeOutcomepCRRCB-IRCB-IIRCB-IIIHR+/HER2-Frequency (%)11%10%52%27%(N=1467)5 yr EFS (95% CI)91% (86-96)93% (89-98)82% (79-85)70% (65-75)10 yr EFS (95% CI)84% (75-93)88% (82-95)71% (67-75)52% (46-58)HR+/HER2+Frequency (%)38%18%35%9%(N=762)5 yr EFS (95% CI)94% (91-97)93% (88-98)78% (73-84)49% (37-65)10 yr EFS (95% CI)91% (86-96)79% (70-90)65% (59-73)42% (29-60)HR-/HER2+Frequency (%)66%11%18%5%(N=550)5 yr EFS (95% CI)93% (90-96)88% (79-97)60% (50-71)45% (30-69)10 yr EFS (95% CI)90% (86-94)84% (74-95)56% (46-68)45% (30-69)HR-/HER2-Frequency (%)41%13%33%13%(N=1293)5 yr EFS (95% CI)92% (90-94)85% (79-91)68% (63-72)28% (21-36)10 yr EFS (95% CI)87% (82-91)80% (72-88)63% (58-68)24% (18-33) Citation Format: Christina Yau, Marieke van der Noordaa, Jane Wei, Marie Osdoit, Fabien Reyal, Anne-Sophie Hamy, Marick Lae, Miguel Martin, Maria del Monte, I-SPY 2 TRIAL Consortium, Judy C Boughey, Rebekah Gould, Jelle Wesseling, Tessa Steenbruggen, Maartje van Seijen, Gabe Sonke, Stephen Edge, Stephen-John Sammut, Elena Provenzano, Jean Abraham, Peter Hall, Ashley Graham, Lorna Mackintosh, David Cameron, Alice Wang, Priyanka Sharma, Kimberly Cole, Lajos Pusztai, Mi-Ok Kim, Laura van ‘t Veer, Laura Esserman, W. Fraser Symmans. Residual cancer burden after neoadjuvant therapy and long-term survival outcomes in breast cancer: A multi-center pooled analysis [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 GS5-01.

Background: In an adaptive randomized trial, when new treatment combinations are being tested, it is important to be able to identify patients who are progressing on treatment so that they can be changed to a different therapeutic regimen. We know that even within the molecularly high risk patients in I-SPY 2, there is considerable variation in biology. In this study, we will present results of using MRI-calculated functional tumor volume (FTV) to identify tumor progression for each breast cancer subtype. Methods: Patients (n=990) enrolled in the I-SPY 2 TRIAL who were randomized to the graduated experimental drug arms or controls from 2010 to 2016 were analyzed. Four MRI exams were performed for each patient: pre-NAC (T0), after 3 weeks of NAC (T1), between regimens (T2), and post-NAC (T3). Functional tumor volume (FTV) was calculated at each exam by summing voxels meeting enhancement thresholds. Tumor progression at T1, T2 or T3 was identified by a positive FTV change relative to T0. Visual inspection was used to exclude false progression due to strong background parenchymal enhancement post-contrast, prominent vessels, motion, or insufficient image quality. pCR was defined as no invasive disease in the breast and lymph nodes. Negative predictive value for pCR was defined as:NPV=number of true non-pCRs / number of patients with MRI assessed tumor progressions, where “true non-pCRs” referred to patients who were non-pCRs at surgery and were assessed as progressors by MRI. The analysis was performed in the full cohort and in sub-cohorts defined by HR and HER2 statuses. Results: Out of 990 patients, 878 had pCR outcome data (pCR or non-pCR, pCR rate = 35%). Total and non-pCR numbers for each subtype, number of patients with tumor progression assessed by MRI at T1, T2, and T3, and NPVs, are shown in Table 1. In the full cohort, the NPV increased consistently over treatment, from T1 (NPV=83%) to T2 (93%), and to T3 (100%). The HER2+ cancer subtypes showed fewer MRI-assessed tumor progressions than HER2- subtypes: e.g. 10/209 (5%) vs. 108/669 (16%) at T1. NPV was 100% for HER2+ subtypes at T1 and T2 except for a single misclassification of a HR- tumor at T1. Only 6 tumor progressors, all HER2- were identified at T3, and all were confirmed at surgery as non-pCRs (NPV=100%). For HR+/HER2-, the NPV increased slightly from 89% at T1 to 91% at T2, while triple negative subtype had a more substantial increase, from 78% to 92%. Conclusions: Our study showed strong association between tumor progressors assessed by MRI with true non-pCRs after NAC. For HER2+ tumors, although MRI progressors are rare, they strongly indicate non-pCR at all treatment time points, while HER2- subtypes show more accurate results later in treatment. We are evaluating MRI change at 6 weeks to determine if that time point is sufficient to predict progressors. Table 1 MRI assessed tumor progression at different treatment time pointN/non-pCRs/%non-pCRMRI assessed tumor progressionT1 (after 3 weeks)T2 (inter-regimen)T3 (post-NAC)NNPV (%)NNPV (%)NNPV (%)Full cohort878/572/65%11883.14192.76100%HR+/HER2-344/280/81%4588.91190.93100%HR+/HER2+134/85/63%610021000N/AHR-/HER2+75/23/31%47521000N/Atriple negative325/184/57%6377.82692.33100% Citation Format: Wen Li, Natsuko Onishi, David C Newitt, Jessica Gibbs, Lisa J Wilmes, Ella F Jones, Bonnie N Joe, Laura S Sit, Christina Yau, A. Jo Chien, Elissa Price, Kathy S Albain, Theresa Kuritza, Kevin Morley, Judy C Boughey, Kathy Brandt, Sadia Choudhery, Amy S Clark, Mark Rosen, Elizabeth S McDonald, Anthony D Elias, Dulcy Wolverton, Kelly Fountain, David M Euhus, Heather S Han, Bethany Niell, Jennifer Drukteinis, Julie E Lang, Janice Lu, Jane L Meisel, Zaha Mitri, Rita Nanda, Donald W Northfelt, Tara Sanft, Erica Stringer-Reasor, Rebecca K Viscusi, Anne M Wallace, Douglas Yee, Rachel Yung, Smita M Asare, Michelle E Melisko, Jane Perlmutter, Hope S Rugo, Richard Schwab, W. Fraser Symmans, Laura J van't Veer, Donald A Berry, Angela DeMichele, Hiroyuki Abe, Deepa Sheth, Kirsten K Edmiston, Erin D Ellis, Richard Ha, Ralph Wynn, Erin P Crane, Charlotte Dillis, Michael Nelson, An Church, Claudine Isaacs, Qamar J Khan, Karen Y Oh, Neda Jafarian, Dae Hee Bang, Christiane Mullins, Stefanie Woodard, Kathryn W Zamora, Haydee Ojeda-Fornier, Pulin Sheth, Linda Hovanessian-Larsen, Mohammad Eghtedari, Michael Spektor, Marina Giurescu, Mary S Newell, Michael A Cohen, Elise Berman, Constance Lehman, William Smith, Kim Fitzpatrick, Marisa H Borders, Wei Yang, Basak Dogan, Sally Goudreau, Thelma Brown, Laura J Esserman, Nola M Hylton. Breast cancer subtype specific association of pCR with MRI assessed tumor volume progression during NAC in the I-SPY 2 trial [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 PD9-04.

Purpose In breast MRI, contrast enhancement of normal fibroglandular tissue is referred to as background parenchymal enhancement (BPE). Hormonal status significantly affects the degree of BPE, potentially due to the association with mammary vascularity and activity1-5. Studies have shown that BPE may be associated with breast cancer survival6, treatment response to neoadjuvant chemotherapy (NAC)7,8 and future breast cancer risk9. In most patients undergoing NAC, BPE is suppressed by the nonspecific anti-proliferative effects of chemotherapy on normal breast and/or ovary5,10. However, some patients exhibit equivalent or even stronger BPE post-NAC compared to pre-NAC. We hypothesized that non-suppressed BPE in post-NAC MRI may be associated with inferior treatment response. This study aimed to investigate the association between BPE suppression and treatment response as defined by pathologic complete response (pCR). Methods This study included patients with stage II/III breast cancer enrolled in the I-SPY 2 TRIAL being treated with standard NAC with or without investigational agents. The whole cohort was split into two subgroups based on hormone receptor status (HR+, n= 536; HR-, n=452). Patients underwent dynamic contrast enhanced MRIs at four time points during NAC: baseline (T0), after 3 weeks of the first regimen (T1), inter-regimen (T2), and pre-surgery (T3). Using in-house software, the contralateral breast parenchyma was automatically segmented for the entire breast volume. Quantitative BPE (qBPE) was calculated as the mean early (~150s post-contrast injection) percent enhancement of the central 50% of the axial slices. A breast radiologist reviewed all exams and excluded those where automated segmentation failed to accurately define tissue. For T1, T2 and T3, BPE was categorized based on the change from T0 as suppressed (qBPE < qBPE[T0]) or non-suppressed (qBPE ≥ qBPE[T0]). Chi-squared test was used to examine the association between BPE suppression and pCR, with p Results HR+ cohort: pCR rates were lower for patients with non-suppressed BPE than those with suppressed BPE at every visit (T1-T3) (Table 1). The difference was statistically significant at T2 (p=0.04) and T3 (p=0.01). Table 1: HR+ cohortpCR rate (%)No. of pCR patientsNo. of non-pCR patientsTotal number of patientsP valueOverall22.8122414536BPE at T1suppressed23.6822663480.45non-suppressed20.532124156BPE at T2suppressed25.7972803770.04*non-suppressed16.01789106BPE at T3suppressed25.7982833810.01*non-suppressed12.5128496 HR- cohort: pCR rates were slightly lower for the non-suppressed BPE group, but no statistically significant association was found (Table 2). Table 2: HR- cohortpCR rate (%)No. of pCR patientsNo. of non-pCR patientsTotal number of patientsP valueOverall44.7202250452BPE at T1suppressed46.81411603010.66non-suppressed44.45265117BPE at T2suppressed48.81441512950.79non-suppressed47.3434891BPE at T3suppressed49.31461502960.94non-suppressed48.9434588 Conclusion In HR+ breast cancer, lack of BPE suppression may indicate inferior treatment response. The contrasting results in HR+ and HR- cohorts are noteworthy in terms of the possible relationship between suppression of normal mammary and ovarian activity and treatment response in HR+ cancer. Reference Radiographics 2014; 34: 234-47. Radiology 1997; 203: 137-44. Radiology 1997; 203: 145-9. Breast J 2005; 11: 236-41. AJR Am J Roentgenol 2015; 204: 669-73. Eur Radiol 2018; 28: 4705-16. Eur Radiol 2016; 26: 1590-6. Transl Oncol 2015; 8: 204-9. J Clin Oncol 2019; : JCO1800378. Radiology 2015; 277: 687-96. Citation Format: Natsuko Onishi, Wen Li, David C. Newitt, Roy Harnish, Jessica Gibbs, Ella F. Jones, Alex Nguyen, Lisa Wilmes, Bonnie N. Joe, Michael J. Campbell, Amrita Basu, Laura J. van’t Veer, Angela DiMichele, Douglas Yee, Donald A. Berry, Kathy S. Albain, Judy C. Boughey, A. Jo Chien, Amy S. Clark, Kirsten K. Edmiston, Anthony D. Elias, Erin D. Ellis, David M. Euhus, Heather S. Han, Claudine Isaacs, Qamar J. Khan, Julie E. Lang, Janice Lu, Jane L. Meisel, Zaha Mitri, Rita Nanda, Donald W. Northfelt, Tara Sanft, Erica Stringer-Reasor, Rebecca K. Viscusi, Anne M. Wallace, Rachel Yung, Michelle E. Melisko, Jane Perlmutter, Hope S. Rugo, Richard Schwab, W. Fraser Symmans, Smita M. Asare, Julie E. Yau, Christina Yau, Laura J. Esserman, Nola M. Hylton. Lack of background parenchymal enhancement suppression in breast MRI during neoadjuvant chemotherapy may be associated with inferior treatment response in hormone receptor positive breast cancer [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 PD9-05.

Background: Strong background parenchymal enhancement (BPE) may cause overestimation in tumor volume measured from dynamic contrast-enhanced (DCE) MRI, which may adversely affect the ability of MR tumor volume to predict treatment outcome for patients undergoing neoadjuvant chemotherapy (NAC). Specifically, an overestimation of tumor volume can result in misclassification of patients with complete pathologic response (pCR) as non-responders, leading to less confidence in MRI prediction. As well, overestimation of extent of disease might lead to more aggressive surgical therapy than necessary. This study investigated whether high BPE in the contralateral breast influences the predictive performance of MRI-measured functional tumor volume (FTV) for patients with locally advanced breast cancer undergoing NAC. Methods: patients (n=990) enrolled in the I-SPY 2 TRIAL who were randomized to the graduated experimental drug arms or controls from 2010 to 2016 were analyzed. Each patient had 4 MRI exams: pre-NAC (T0), after 3 weeks of NAC (T1), between NAC regimens (T2), and post-NAC (T3). FTV was calculated at each MRI exam by summing voxels meeting enhancement thresholds. Background parenchymal enhancement (BPE) in the contralateral breast was calculated automatically as mean percentage enhancement on the early (nominal 150sec post-contrast) image in the fibroglandular tissue segmented from 5 continuous axial slices centered in the inferior-to-superior stack. For each treatment time point, patients having both FTV and BPE measurements were included in the analysis. The area under the ROC curve (AUC) was estimated as the association between FTV and pCR at T1, T2, and T3. The analysis was conducted in the full patient cohort and in sub-cohorts defined by hormone receptor (HR) and HER2 status. In each patient cohort, a cut-off BPE value was selected to classify patients with high vs. low BPE by testing AUCs estimated with low-BPE patients reached maximum when the cut-off value varied from median to maximum in steps of 10%. Results: Out of 990 patients, 878 had pCR outcome data (pCR or non-pCR, pCR rate = 35%). Table 1 shows the number of patients, pCR rate, and AUC of FTV for predicting pCR using all patients available vs. a subset patients with low BPE (< BPE cut-off). In the full cohort, AUC increased slightly across all time points after patients with high BPE were removed. In the HR+/HER2- subtype, AUC increased at T1 after removal of cases with high BPE (0.65 vs. 0.71). For HR-/HER2+, AUC increased substantially after removal of high BPE cases (0.65 to 0.86 at T1, 0.71 to 0.87 at T2, and 0.71 to 0.89 at T3), with greater improvement at the early time point (T1) compared to later time points (T2 and T3). Only a slight improvement in the AUC was observed in the HR+/HER2+ and HR-/HER2- subtypes across all time points. Conclusions: High background parenchymal enhancement adversely affected the predictive performance of functional tumor volume measured by DCE-MRI, at early treatment time point for HR+/HER2- and across all time points for HR-/HER2+ cancer subtype. The adverse effect might be offset using subtype-optimized enhancement threshold in calculating functional tumor volume. Table 1 Effect of BPE on the prediction of pCR using FTV at various treatment time pointsT1T2T3npCR rateAUCBPE cut-offnpCR rateAUCBPE cut-offnpCR rateAUCBPE cut-offFullAll64734%0.662762334%0.701761134%0.6925Subset45334%0.6831133%0.7230534%0.72HR+/HER2-All26218%0.651924918%0.718225518%0.7519Subset13118%0.7124818%0.7120419%0.76HR+/HER2+All10636%0.642110538%0.62269634%0.7120Subset5332%0.668438%0.665740%0.73HR-/HER2+All5175%0.65204774%0.71204973%0.7116Subset3073%0.862871%0.872475%0.89HR-/HER2-All22842%0.682822243%0.751821143%0.6916Subset15940%0.7111137%0.7810540%0.75 Citation Format: Wen Li, Natsuko Onishi, David C Newitt, Roy Harnish, Ella F Jones, Lisa J Wilmes, Jessica Gibbs, Elissa Price, Bonnie N Joe, A. Jo Chien, Donald A Berry, Judy C Boughey, Kathy S Albain, Amy S Clark, Kirsten K Edmiston, Anthony D Elias, Erin D Ellis, David M Euhus, Heather S Han, Claudine Isaacs, Qamar J Khan, Julie E Lang, Janice Lu, Jane L Meisel, Zaha Mitri, Rita Nanda, Donald W Northfelt, Tara Sanft, Erica Stringer-Reasor, Rebecca K Viscusi, Anne M Wallace, Douglas Yee, Rachel Yung, Michelle E Melisko, Jane Perlmutter, Hope S Rugo, Richard Schwab, W. Fraser Symmans, Laura J van't Veer, Christina Yau, Smita M Asare, Angela DeMichele, Sally Goudreau, Hiroyuki Abe, Deepa Sheth, Dulcy Wolverton, Kelly Fountain, Richard Ha, Ralph Wynn, Erin P Crane, Charlotte Dillis, Theresa Kuritza, Kevin Morley, Michael Nelson, An Church, Bethany Niell, Jennifer Drukteinis, Karen Y Oh, Neda Jafarian, Kathy Brandt, Sadia Choudhery, Dae Hee Bang, Christiane Mullins, Stefanie Woodard, Kathryn W Zamora, Haydee Ojeda-Fornier, Mohammad Eghedari, Pulin Sheth, Linda Hovanessian-Larsen, Mark Rosen, Elizabeth S McDonald, Michael Spektor, Marina Giurescu, Mary S Newell, Michael A Cohen, Elise Berman, Constance Lehman, William Smith, Kim Fitzpatrick, Marisa H Borders, Wei Yang, Basak Dogan, Laura J Esserman, Nola M Hylton. The effect of background parenchymal enhancement on the predictive performance of functional tumor volume measured in MRI [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 P6-02-01.

Cancer research is moving at a startling pace, most particularly with the identification and characterization of molecular signatures and biomarkers that will be used for prevention, early detection, diagnosis, treatment, prediction, and prognostication. The goal of cancer therapy is to match the right treatment with the right patient. To this end, several clinical trials are now employing patient stratification using “Massive Parallel Sequencing” or informally called “Next -Generation Sequencing” (NGS) to identify clinically actionable targets in real time.

The precision medicine (PM) revolution is well underway, and next-generation sequencing (NGS) is helping take cancer treatment to new levels. Finding actionable targets in real time is now a reality, and data from several clinical trials based on identified molecular alterations can be assessed and can help address the question of whether personalized treatment based on genomics produces superior survival outcomes compared with unselected treatment. Targeted treatment-based clinical trials have shown benefit in molecular subgroups of patients. Of further interest, it appears that genomics and immunotherapy are coupled to each other, since the immune system recognizes neo-antigens produced by the mutanome. Hence, some of the most important markers predicting response to immunotherapy are genomic markers, PDL1 amplification (for PD-1/PD-L1 checkpoint inhibitors), and tumor mutational burden. However, a number of challenges remain to be addressed if the use of molecular profiling-guided therapy in PM-designed clinical trials is to become the standard on which cancer treatment is based. In this chapter, we explore what it will take for PM trials that use molecular profiling as part of the eligibility criteria to be successful and the remaining hurdles that need to be overcome.

Objective Different molecular subtypes of triple-negative breast cancer (TNBC) have previously been identified through analysis of gene expression profiles. The luminal androgen receptor (LAR) subtype has been shown to have a lower rate of pathologic complete response to neoadjuvant chemotherapy than other TNBC subtypes. The purpose of this study was to determine if the imaging features of TNBCs differ by AR (androgen receptor) status, which is a surrogate immunohistochemical (IHC) marker for the chemoresistant LAR subtype of TNBC. Materials and methods This sub-study was part of a clinical trial in patients with stage I-III TNBC who were prospectively monitored for response while receiving neoadjuvant systemic therapy (NAST) at a single comprehensive cancer center. This interim imaging analysis included 144 patients with known AR status measured by IHC. AR-positive (AR+) tumors were defined as those in which at least 10% of tumor cells had positive nuclear AR staining. Two experienced, fellowship-trained breast radiologists who were blinded to the IHC results retrospectively reviewed and reached consensus on all imaging studies for the index lesion (i.e., mammogram, ultrasound, and breast magnetic resonance imaging). The index lesion for each patient was reviewed and described according to the fifth edition of the Breast Imaging Reporting and Data System lexicon. Logistic regression modeling was used to identify imaging features predictive of AR status. p ≤ 0.05 was considered statistically significant. Results Univariate logistic regression models for AR status showed that AR+ TNBC was significantly associated with heterogeneously dense breast composition on mammography (p = 0.02), mass with calcifications (p = 0.05), irregular mass shape on mammography (p = 0.03), and irregular mass shape on sonography (p = 0.003). Multivariate logistic regression models for AR status showed that AR+ TNBC was significantly associated with heterogeneously dense breast composition on mammography (p = 0.01), high mass density on mammography (p = 0.003), and irregular mass shape on sonography (p = 0.0004). Conclusion The imaging features of TNBCs differ by AR status. Multimodality breast imaging may help identify the LAR subtype of TNBC, which has been shown to be a subtype that is relatively resistant to neoadjuvant chemotherapy.

Jeffrey S Ross1†, W Fraser Symmans2, Lajos Pusztai3 & Gabriel N Hortobagyi3 †Author for correspondence 1Albany Medical College, Department of Pathology and Laboratory Medicine, 47 New Scotland Avenue, Albany, NY 12208, USA Tel.: +1 518 262 5461; Fax: +1 518 262 3663; E-mail: rossj@mail.amc.edu 2The University of Texas M.D. Anderson Cancer Center, Division of Pathology, Houston, TX, USA 3The University of Texas M.D. Anderson Cancer Center, Division of Breast Medical Oncology, Houston, TX, USA “Rapid expansion of future novel diagnostics designed to personalize breast cancer care can easily blind us to the unmet needs for improving the accuracy and reliability of tests that are already in common daily clinical practice.”

Advances in the surgical management of the axilla in patients treated with neoadjuvant chemotherapy, especially those with node positive disease at diagnosis, have led to changes in practice and more judicious use of axillary lymph node dissection that may minimize morbidity from surgery. However, there is still significant confusion about how to optimally manage the axilla, resulting in variation among practices. From the viewpoint of drug development, assessment of response to neoadjuvant chemotherapy remains paramount and appropriate assessment of residual disease—the primary endpoint of many drug therapy trials in the neoadjuvant setting—is critical. Therefore decreasing the variability, especially in a multicenter clinical trial setting, and establishing a minimum standard to ensure consistency in clinical trial data, without mandating axillary lymph node dissection, for all patients is necessary. The key elements which include proper staging and identification of nodal involvement at diagnosis, and appropriately targeted management of the axilla at the time of surgical resection are presented. The following protocols have been adopted as standard procedure by the I-SPY2 trial for management of axilla in patients with node positive disease, and present a framework for prospective clinical trials and practice.

Magnetic resonance imaging (MRI) captures the three dimensional way in which tumors are organized in the breast, defined as imaging phenotypes in the I-SPY 1 trial. We developed a gene set based on the way in which breast epithelial cells aggregate and organize in three dimensional cultures. We investigated whether these organizational genes correspond to the imaging phenotypes. MRI phenotypes have been shown to correspond to the degree of response to neoadjuvant chemotherapy, and are used to predict the ability to achieve breast conservation treatment (Mukhtar et al, Ann Surg Oncol, 20: 3823–3830, 2013). We hypothesized that the molecular profile accompanying phenotypic changes occurring during the organization process of non-malignant acini may explain the molecular basis of MRI tumor phenotypes. We have developed prediction models for MRI phenotypes based on expression profiles identified during the organization process of non-malignant breast epithelial cells in three-dimensional laminin-rich extracellular matrix (Fournier et al. Cancer Res, 66, 7095-102, 2006). We analyzed a subset of 324 organizational genes in 147 samples from stage II-III breast cancer in the I-SPY 1 trial cohort with Agilent microarrays and MRI annotation (CALGB 150007/150012; ACRIN 6657). MRI phenotype of the index lesion was assessed by the site radiologists using the following radiographic criteria: A) well defined unicentric mass; B) well-defined multilobulated mass; C) area enhancement with nodularity; D) area enhancement without nodularity; E) Septal spreading. The distribution of phenotypes in I-SPY 1 was: A: 16%, B: 33%, C: 30%, D: 15%, E: 7%. We developed predictors for MRI phenotypes dichotomized as either "well-defined" (A and B) or "non-well-defined" (C, D and E). Using patent-pending algorithms we selected a subset of the organizational genes with the greatest predictive power to identify MRI "well-defined" phenotypes in the I-SPY 1 cohort. Then prediction models were developed using the 50 top ranking genes and logistic regression methods. We followed Monte-Carlo cross validation method to make sure that the performance of a model is not a result of over-fitting the model to the sample data using separate datasets to support the modeling. The samples were randomly partitioned 10,000 times in a 85% training, and 15% test ratio to test models performance. The resulting performance of predictive models in the test set had an average classification accuracy of greater than 80% (ROC statistics AUC>0.8) using gene expression measurements from between 16 to 20 genes. For random lists of 16-20 genes, the accuracy was approximately 50% (ROC AUC∼0.5). Amongst the MRI models were several genes that are known to regulate key cellular pathways such as cell division, metabolism, and migration using MetaCore pathway analysis. Taken together, the results suggest that the MRI phenotypes may be a manifestation of the organization genes that determine behavior in three dimensional culture. Future research includes confirming these results using an independent dataset, defining the potential drivers of MRI phenotypes, and determining if putative drivers provide a key contribution to tumor subtypes. Citation Format: Marcia V Fournier, Alan Derr, Alex Margulis, Kevin Reid, Sara Brumbaugh, Richard Anderson, W Fraser Symmans, Laura Esserman, Nola Hylton. Gene expression profiles accompanying phenotypic changes during non-malignant breast epithelial cells acini formation to explain MRI phenotypes [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 P3-06-19.

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 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.

A subset of early stage estrogen receptor (ER)-positive breast cancers considered “high risk” for recurrence with endocrine therapy alone by current genomic prognostic predictors, such as Oncotype DX, is no longer high risk after receiving adjuvant chemotherapy. We hypothesized that a recently described gene expression-based outcome predictor adjuvant chemotherapy and endocrine therapy sensitivity (ACES) could re-stratify these patients into high and low risk groups for relapse when treated with both chemo- and endocrine therapies. ACES involves four separate modules (endocrine sensitivity, chemotherapy sensitivity, chemotherapy resistance, and survival prediction) that yield a prediction for good or poor outcome with current standard of care multimodality therapy. ACES was applied to Affymetrix gene expression data from 2 retrospectively collected ER-positive and HER2-negative patient cohorts that were uniformly treated with adjuvant endocrine and chemotherapy (n = 250). Each sample was first risk stratified by a genomic surrogate of Oncotype DX, and the high risk patients (n = 76) were re-stratified by ACES. Recurrence-free survival (RFS) was evaluated with ACES risk categories. The Oncotype DX high risk but ACES good prognosis patients (n = 24, 32 %) had an RFS of 95 % compared to 76 % in the poor prognosis group (n = 52; log-rank p = 0.033) at 5 years. ACES risk category remained an independent predictor in multivariate analysis after adjusting for age, T-stage, and lymph node involvement at diagnosis (hazard ratio 0.15; p = 0.072). Tertiary risk prediction that takes into account chemotherapy and endocrine sensitivity, and baseline prognosis may help identify high risk ER-positive patients who have excellent survival after chemotherapy.

Background Although several randomised trials in patients with triple-negative breast cancer have shown that the addition of carboplatin, with or without poly(ADP-ribose) polymerase (PARP) inhibitors, to neoadjuvant chemotherapy increases the likelihood of achieving a pathological complete response, the use of these therapies in this setting has remained controversial. The BrighTNess trial was designed to assess the addition of the PARP inhibitor veliparib plus carboplatin or carboplatin alone to standard neoadjuvant chemotherapy in triple-negative breast cancer. Methods We did a phase 3, randomised, double-blind, placebo-controlled trial (BrighTNess) across 145 sites in 15 countries. Patients aged 18 years and older with previously untreated histologically or cytologically confirmed clinical stage II-III triple-negative breast cancer, who were candidates for potentially curative surgery and had an Eastern Cooperative Oncology Group performance status of 0 or 1, were randomly assigned (2:1:1) by an interactive response technology system via permuted blocks (block size of four) within strata to receive one of three segment 1 regimens: paclitaxel (80 mg/m(2) intravenously weekly for 12 doses) plus carboplatin (area under the curve 6 mg/mL per min, intravenously every 3 weeks, for four cycles) plus veliparib (50 mg orally, twice a day); paclitaxel plus carboplatin plus veliparib placebo (twice a day); or paclitaxel plus carboplatin placebo (every 3 weeks for four cycles) plus veliparib placebo. Following segment 1, all patients were assigned to segment 2 in which they received doxorubicin and cyclophosphamide every 2-3 weeks for four cycles. Randomisation for segment 1 was stratified by germline BRCA mutation status, nodal stage, and planned schedule of doxorubicin and cyclophosphamide administration. The primary endpoint was pathological complete response in breast and lymph nodes as determined by site pathologists following completion of neoadjuvant therapy. Efficacy analyses were done by intention to treat and safety analyses included all patients who received at least one dose of study treatment. These are the first results of an ongoing clinical trial; the data cutoff for the analyses presented was Dec 8, 2016. This study is registered with ClinicalTrials.gov, number NCT02032277. Findings Between April 4, 2014, and March 18, 2016, 634 patients were randomly assigned: 316 to paclitaxel plus carboplatin plus veliparib, 160 to paclitaxel plus carboplatin, and 158 to paclitaxel alone. The proportion of patients who achieved a pathological complete response was higher in the paclitaxel, carboplatin, and veliparib group than in patients receiving paclitaxel alone (168 [53%] of 316 patients vs 49 [31%] of 158, p

Purpose To determine the long-term prognosis in each phenotypic subset of breast cancer related to residual cancer burden (RCB) after neoadjuvant chemotherapy alone, or with concurrent human epidermal growth factor receptor 2 (HER2)–targeted treatment. Methods We conducted a pathologic review to measure the continuous RCB index (wherein pathologic complete response has RCB = 0; residual disease is categorized into three predefined classes of RCB index [RCB-I, RCB-II, and RCB-III]), and yp-stage of residual disease. Patients were prospectively observed for survival. Three patient cohorts received paclitaxel (T) followed by fluorouracil, doxorubicin, and cyclophosphamide (T/FAC): original development cohort (T/FAC-1), validation cohort (T/FAC-2), and independent validation cohort (T/FAC-3). Another validation cohort received FAC chemotherapy only, and a fifth cohort received concurrent trastuzumab (H) with sequential paclitaxel and fluorouracil, epirubicin, and cyclophosphamide (FEC; H+T/FEC). Phenotypic subsets were defined by hormone receptor (HR) and HER2 status at diagnosis, classified as HR-positive/HER2-negative, HER2-positive (HR-negative/HER2-positive or HR-positive/HER2-positive), or triple receptor–negative. Relapse-free survival estimates were determined from Kaplan-Meier analysis and compared using the log-rank test. Results Five cohorts (T/FAC-1 [n = 219], T/FAC-2 [n = 262], T/FAC-3 [n = 342], FAC [n = 132], and H+T/FEC [n = 203]) had median event-free follow-up of 13.5, 9.1, 6.8, 16.4, and 7.1 years, respectively. Continuous RCB index was prognostic within each phenotypic subset, independent of other clinical-pathologic variables. RCB classes stratified prognostic risk overall, within each phenotypic subset, and within yp-stage categories. Estimates of 10-year relapse-free survival rates in the four RCB classes (pathologic complete response, RCB-I, RCB-II, and RCB-III) were 86%, 81%, 55%, and 23% for triple receptor–negative; 83%, 97%, 74%, and 52% for HR-positive/HER2-negative in the combined T/FAC cohorts; and 95%, 77%, 47%, and 21% in the H+T/FEC cohort. Conclusion RCB was prognostic for long-term survival after neoadjuvant chemotherapy in all three phenotypic subsets of breast cancer. Our institutional findings should be externally validated.

The main objective is to accurately identify the sites of primary tumor bed (that may or may not contain residual cancer cells) and any residual lymph node metastases, so that the extent of residual cancer can be measured and reported. Of course, the tumor bed can become very subtle after effective neoadjuvant chemotherapy, and improving treatments make this ever more likely. Posttreatment residual disease is often detected by light palpation of the sliced breast specimen even more easily than it is observed by visual inspection. Sometimes that is in an area of ill-defined fibrosis. Fortunately, the communication between pathologists, surgeons, and radiologists has greatly improved and, combined with innovations in preoperative localization, has greatly improved the precision of pathologic evaluation.

Background. The aim of this study was to assess the frequency of potentially actionable genomic alterations in breast cancer that could be targeted with approved agents or investigational drugs in clinical trials using a next-generation sequencing-based genomic profiling assay performed in a Clinical Laboratory Improvement Amendments-certified and College of American Pathologists-accredited commercial laboratory. Methods. Fifty-one breast cancers were analyzed, including primary tumor biopsies of 33 stage I–II and 18 stage IV cancers (13 soft tissue, 3 liver, and 2 bone metastases). We assessed 3,230 exons in 182 cancer-related genes and 37 introns in 14 genes often rearranged in cancer for base substitutions, indels, copy number alterations, and gene fusions. The average median sequencing depth was 1,154×. Results. We observed 158 genomic alterations in 55 genes in 48 of 51 (94%) tumors (mean 3.1, range 0–9). The average number of potentially therapeutically relevant alterations was similar in primary (1.6, range 0–4) and in heavily pretreated metastatic cancers (2.0, range 0–4) (p = .24). The most common actionable alterations were in PIK3CA (n = 9, phosphatidylinositol 3-kinase [PI3K]/mammalian target of rapamycin [mTOR] inhibitors), NF1 (n = 7, PI3K/mTOR/mitogen-activated protein kinase inhibitors), v-akt murine thymoma viral oncogene homolog 1-3 (n = 7, PI3K/mTOR/AKT inhibitors), BRCA1/2 (n = 6, poly[ADP-ribose] polymerase inhibitors), and CCND1,2 and CCNE (n = 8)/cycline dependent kinase (CDK)6 (n = 1) (CDK4/6 inhibitors), KIT (n = 1, imatinib/sunitinib), ALK (n = 1, crizotinib), FGFR1,2 (n = 5, fibroblast growth factor receptor inhibitors), and EGFR (n = 2, epidermal growth factor receptor inhibitors). Our sequencing assay also correctly identified all six cases with HER2 (ERBB2) amplification by fluorescence in situ hybridization when tumor content was adequate. In addition, two known activating HER2 mutations were identified, both in unamplified cases. Conclusion. Overall, 84% of cancers harbored at least one genomic alteration linked to potential treatment options. Systematic evaluation of the predictive value of these genomic alterations is critically important for further progress in this field.

Tissue handling can alter global gene expression potentially affecting the analytical performance of genomic signatures, but such effects have not been systematically evaluated.Tissue samples from 11 previously untreated breast tumors were minced and aliquots were either snap frozen or placed in RNAlater immediately or after 20, 40, 60, 120 or 180 min at room temperature. RNA was profiled on Affymetrix HG-U133A arrays. We used probe-set-wise hierarchical models to evaluate the effect of preservation method on transcript expression and linear mixed effects models to assess the effect of cold ischemic delay on the expression of individual probe sets. Gene set enrichment analysis identified pathways overrepresented in the affected transcripts. We combined the levels of 41 most sensitive transcripts to develop an index of ischemic stress.Concordance in global gene expression between the baseline and 40 min delay was higher for samples preserved in RNAlater (average concordance correlation coefficient CCC = 0.92 compared to 0.88 for snap frozen). Overall, 481 transcripts (3%) were significantly affected by the preservation method, most of them involved in processes important in cancer. Prolonged cold ischemic delay of up to 3 h induced marginal global gene expression changes (average CCC = 0.90 between baseline and 3 h delay). However 41 transcripts were significantly affected by cold ischemic delay. Among the induced transcripts were stress response genes, apoptotic response genes; among the downregulated were genes involved in metabolism, protein processing and cell cycle regulation. An index combining the expression levels of these genes was proportional to the cold ischemic delay.Prolonged cold ischemia induces significant transcriptional changes in a small subset of transcripts in the tissue. Furthermore, the expression level of about 3% of the transcripts is affected by the preservation method. These sensitive transcripts should not be included in genomic signatures for more reliable analytical performance.

Background: Differences in gene or protein expression patterns between breast cancers according to race/ethnicity and cancer subtype. Methods: Transcriptional profiling was performed using Affymetrix HG-U133A platform in 376 patients and reverse phase protein array analysis (RPPA) was done for 177 proteins in 255 patients from a separate cohort. Unsupervised clustering was conducted, as well as supervised comparison by race and tumor subtype. Standard statistical methods, BRB-Array tools, and Ingenuity Pathways software packages were used to analyze the data. Results: Median age was 50 years in both the cohorts. In the RPPA cohort, 54.5% of the tumors were hormone receptor–positive (HR-positive), 20.7% HER2-positive, and 24.71% triple-negative (TNBC). One hundred and forty-seven (57.6%), 47 (18.43%), and 46 (18.1%) of the patients were White, Hispanic, and Black, respectively. Unsupervised hierarchical clustering of the protein expression data showed no distinct clusters by race (P values were 0.492, 0.489, and 0.494 for the HR-positive, HER2-positive, and TNBC tumors respectively). In the gene expression cohort, 54.2% of the tumors were HR-positive, 16.5% HER2-positive, and 29.3% TNBC. Two hundred and sixteen (57.5%), 111 (29.52%), and 32 (8.52%) patients were White, Hispanic, and Black, respectively. No probe set with a false discovery rate (FDR) of Conclusions: We did not detect a significant variation in RNA or protein expression comparing different race/ethnicity groups of women with breast cancer. Impact: More research on the complex network of factors that result in outcomes differences among race/ethnicities is needed. Cancer Epidemiol Biomarkers Prev; 23(2); 316–23. ©2013 AACR.

Background: Patients with triple-negative breast cancers (TNBCs) develop chemotherapeutic resistance and have a high mortality rate. Classification of TNBCs based on molecular profiling has enabled the identification of one subtype that is particularly enriched for cancer stem cells (CSCs) and genes involved in epithelial-mesenchymal transition (EMT) pathways. The EMT pathways, as well as CSCs, have been independently associated with chemo-resistance. We have previously demonstrated that cancer cells can acquire CSC properties via the activation of EMT. The FOXC2 transcription factor is a critical mediator for EMT/CSC properties as well as metastatic competence. Activated p38 MAP kinase is also high in EMT/CSC-enriched TNBCs and is important for FOXC2 protein stability, CSC and EMT properties as well as metastasis. We hypothesize that the inhibition of p38 MAP kinase will increase the sensitivity of TNBCs to chemotherapeutic drugs and in combination, it will help reduce tumor recurrence. Methods: Three clinical candidate p38 inhibitors (LY2228820, VX-702, and PH-797804) were tested next to SB203580, which has shown great efficacy in our pre-clinical models. Effects of p38 inhibition were measured using sphere assay, CD44hi/CD24lo profiles and their effect on FOXC2 protein levels using cells lines as well as cells isolated from PDX. To identify drugs that work in combination with p38 inhibitor, cancer cells were pre-treated with p38 inhibitors and then co-treated with p38 inhibitor as well as NCI Approved Oncology/Custom Clinical Collections of 256 approved drugs and the Broad Informer Collection of about 400 mechanistic compounds. Results: Inhibition of p38 significantly impaired stem cell properties in a dose-dependent manner. P38 inhibitor worked in combination with Gemcitabine or Epirubicin (currently used for TNBC treatment) and reduced the TNBC cell proliferation. Among PDX samples capable of forming spheres, p38 inhibition reduced their stemness in a dose-dependent manner. P38 inhibition also decreased expression of the stemness marker FOXC2 and increased the expression of the epithelial marker E-cadherin. Conclusion: P38 inhibition is an effective way to inhibit EMT/CSC properties and it can work in combination with other agents to increase sensitivity in TNBCs. Acknowledgments: This work was supported by CPRIT-MIRA RP-160710 (SAM, WFS). Citation Format: Petra den Hollander, Shruti Shah, Xinhui Zhou, Abena Redwood, Shi-Rong Cai, Mary Sobieski, David Brunell, Clifford Stephan, Peter Davies, Helen Piwnica-Worms, Stacy Moulder, W Fraser Symmans, Sendurai A. Mani. Overcoming therapy resistance in stem cell-rich triple negative breast cancer through p38 MAP kinase inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4669.

Background: I-SPY2 is a multicenter, Phase II trial using response-adaptive randomization within biomarker subtypes to evaluate novel agents as neoadjuvant therapy for high-risk at least T2N0 breast cancer. The primary endpoint is pathologic complete response (pCR) at surgery. The goal is to identify regimens that have ≥ 85% Bayesian predictive probability of success in a 300-patient phase 3 neoadjuvant trial defined by hormone-receptor (HR) and HER2 status, and MammaPrint (MP). Regimens may leave the trial for futility (< 10% probability of success), maximum sample size accrual (with probability of success ≥ 10% and < 85%), or as recommended by the independent DSMB. For HER2- subjects the control arm is weekly paclitaxel x12 then doxorubicin & cyclophosphamide (AC) q2-3 weeks x4. For this arm, paclitaxel was omitted and replaced with maximum tolerated dose PARPi talazoparib with synergy dosed irinotecan (25mg/m2). Paclitaxel could be given adjuvantly for these subjects and non-responding subjects could be taken off of experimental therapy. Methods: Women with tumors ≥ 2.5cm were eligible for screening. MP low/HR+ tumors were ineligible. MRI scans (baseline, 3 cycles after start of therapy, prior to AC, and prior to surgery) were used in a longitudinal statistical model to predict pCR for individual patients. Talazoparib was given at 1mg daily with 25mg/m2 irinotecan q2wks. Analysis was intention to treat. Subjects who switched to non-protocol therapy count as non-pCR. Subjects on experimental therapy at time of arm closure are non-evaluable. Talazoparib/irinotecan (TI) was open only to HER2- tumors and eligible for graduation in 3 of 10 pre-defined signatures: HER2-, HR+HER2- and HR-/HER2-. Results: TI did not meet criteria for graduation and was stopped at the recommendation of the DSMB based on expectations of limited activity beyond that seen with standard treatment. Maximum sample size had been reached at the time of this recommendation and subjects currently receiving TI were allowed to continue or change to standard therapy. Exploratory “as treated” analysis for response in gBRCA mutation carriers showed 6/10 gBRCA carriers attained a pCR in the TI arm. Except for 1 patient these gBRCA pCR subjects had >90% tumor reduction by MRI after TI and prior to AC (range: 68-96%). In the TI arm pCR rates were also higher in subjects with a PARPi7-High/MP2 gene expression signature (0.344 vs 0.146). Expected differences in toxicity were seen between arms including g3/4 peripheral neuropathy on control therapy which included paclitaxel (2.6% vs none) and g3/4 neutropenia with TI (30.2% vs 8.2%). Notably gBRCA mutation carriers receiving TI had higher rates of g3/4 neutropenia (60% vs 25.9%). Conclusion: The I-SPY2 study finds the probability that investigational regimens will be successful in a Phase III neoadjuvant trial; TI did not reach the efficacy threshold of 85% probability of success in Phase III in any of the 3 signatures. However by adding talazoparib with synergy dosed irinotecan we were able to omit paclitaxel and observe similar estimated pCR rates. This informs current work to evolve the I-SPY2 trial design to reduce toxicity without compromising outcomes and develop successful combinations targeted to biology, including DNA repair deficiency. Citation Format: Richard Schwab, Amy S. Clark, Christina Yau, Nola Hylton, Wen Li, Denise Wolfe, A Jo Chien, Anne M. Wallace, Andres Forero-Torres, Erica Stringer-Reasor, Rita Nanda, Nora Jaskowiak, Judy Boughey, Tufia Haddad, Heather S. Han, Catherine Lee, Kathy Albain, Claudine Isaacs, Anthony D. Elias, Erin D. Ellis, Payal Shah, Julie E. Lang, Janice Lu, Debasish Tripathy, Kathleen Kemmer, Douglas Yee, Barbara Haley, Melanie Majure, Erin Roesch, Christos Vaklavas, Cheryl Ewing, Teresa Helsten, W Fraser Symmans, Jane Perlmutter, Hope S. Rugo, Michelle Melisko, Amy Wilson, Ruby Singhrao, Laura van 't Veer, Angela DeMichele, Smita Asare, Don Berry, Laura J. Esserman. Evaluation of talazoparib in combination with irinotecan in early stage, high-risk HER2 negative breast cancer: Results from the I-SPY 2 TRIAL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT136.

We examined estrogen receptor (ER) mRNA expression and molecular subtypes in stage I-III breast cancers that are progesterone receptor (PR) positive but ER and HER2 negative by immunohistochemistry (IHC) or fluorescent in situ hybridization. The ER, PR, and HER2 status was determined by IHC as part of routine clinical assessment (N = 501). Gene expression profiling was done with the Affymetrix U133A gene chip. We compared expressions of ESR1 and MKI67 mRNA, distribution of molecular subtypes by the PAM50 classifier, the sensitivity to endocrine therapy index, and the DLDA30 chemotherapy response predictor signature among ER/PR-positive (n = 223), ER-positive/PR-negative (n = 73), ER-negative/PR-positive (n = 20), and triple-negative (n = 185) cancers. All patients received neoadjuvant chemotherapy with an anthracycline and taxane and had adjuvant endocrine therapy only if ER or PR > 10 % positive. ESR1 expression was high in 25 % of ER-negative/PR-positive, in 79 % of ER-positive/PR-negative, in 96 % of ER/PR-positive, and in 12 % of triple-negative cancers by IHC. The average MKI67 expression was significantly higher in the ER-negative/PR-positive and triple-negative cohorts. Among the ER-negative/PR-positive patients, 15 % were luminal A, 5 % were Luminal B, and 65 % were basal like. The relapse-free survival rate of ER-negative/PR-positive patients was equivalent to ER-positive cancers and better than the triple-negative cohort. Only 20-25 % of the ER-negative/PR-positive tumors show molecular features of ER-positive cancers. In this rare subset of patients (i) a second RNA-based assessment may help identifying the minority of ESR1 mRNA-positive, luminal-type cancers and (ii) the safest clinical approach may be to consider both adjuvant endocrine and chemotherapy.

In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67's value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study.Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays-one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided.Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation.Substantial variability in Ki67 scoring was observed among some of the world's most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited.

Taxanes are among the drugs most commonly used for preoperative chemotherapy for breast cancer. Taxanes induce mitotic arrest and subsequent apoptosis. The spindle-assembly checkpoint (SAC) is known to be activated during mitosis, along with cyclin-dependent kinase-1 (CDK1), and is required for taxane-induced cell death. We hypothesized that CDK1 activity predicts response to taxane-containing chemotherapy. This study included breast cancer patients who received preoperative chemotherapy- taxane-containing treatment followed by anthracycline-based treatment-and then underwent surgery. Before starting taxane-containing chemotherapy, patients underwent fine-needle aspiration biopsy, and the biopsy samples were incubated in paclitaxel solution to measure CDK activity. Clinical were evaluated after taxane therapy, and pathological resposes were evaluated after completion of all preoperative chemotherapy. Thirty five patients were eligible for analysis of clinical response to taxane-containing therapy. Twenty-six patients had taxane-sensitive and 9 taxane-resistant tumors. Using a cut-off of CDK activity determined by the ROC analysis, patients were classified into SAC function and dysfunction groups. Univariate logistic regression analysis with clinicopathologic parameters showed that only CDK-based SAC functionality was significantly correlated with clinical response (P =0.017). No significant correlation was observed between SAC functionality and pathologic response. CDK-based SAC functionality significantly predicted clinical response (P =.0072, overall agreement = 71.4%), and this is a unique mechanism-based marker for predicting taxane chemosensitivity. Further, large prospective study is needed to determine CDK-based SAC functionality could be developed as a predictive biomarker.

Purpose: The clinical relevancy of the 7-subtype classification of triple-negative breast cancer (TNBC) reported by Lehmann and colleagues is unknown. We investigated the clinical relevancy of TNBC heterogeneity by determining pathologic complete response (pCR) rates after neoadjuvant chemotherapy, based on TNBC subtypes. Experimental Design: We revalidated the Lehmann and colleagues experiments using Affymetrix CEL files from public datasets. We applied these methods to 146 patients with TNBC with gene expression microarrays obtained from June 2000 to March 2010 at our institution. Of those, 130 had received standard neoadjuvant chemotherapy and had evaluable pathologic response data. We classified the TNBC samples by subtype and then correlated subtype and pCR status using Fisher exact test and a logistic regression model. We also assessed survival and compared the subtypes with PAM50 intrinsic subtypes and residual cancer burden (RCB) index. Results: TNBC subtype and pCR status were significantly associated (P = 0.04379). The basal-like 1 (BL1) subtype had the highest pCR rate (52%); basal-like 2 (BL2) and luminal androgen receptor had the lowest (0% and 10%, respectively). TNBC subtype was an independent predictor of pCR status (P = 0.022) by a likelihood ratio test. The subtypes better predicted pCR status than did the PAM50 intrinsic subtypes (basal-like vs. non basal-like). Conclusions: Classifying TNBC by 7 subtypes predicts high versus low pCR rate. We confirm the clinical relevancy of the 7 subtypes of TNBC. We need to prospectively validate whether the pCR rate differences translate into long-term outcome differences. The 7-subtype classification may spur innovative personalized medicine strategies for patients with TNBC. Clin Cancer Res; 19(19); 5533–40. ©2013 AACR.

DNA repair pathways can enable tumor cells to survive DNA damage induced by chemotherapy and thus provide prognostic and/or predictive value. We evaluated Affymetrix gene expression profiles for 145 DNA repair genes in untreated breast cancer (BC) patients (n = 684) and BC patients treated with regimens containing neoadjuvant taxane/anthracycline (n = 294) or anthracycline (n = 210). We independently assessed estrogen receptor (ER)-positive/HER2-negative, HER2-positive, and ER-negative/HER2-negative subgroups for differential expression, bimodal distribution, and the prognostic and predictive value of DNA repair gene expression. Twenty-two genes were consistently overexpressed in ER-negative tumors, and five genes were overexpressed in ER-positive tumors, but no differences in expression were associated with HER2 status. In ER-positive/HER2-negative tumors, the expression of nine genes (BUB1, FANCI, MNAT1, PARP2, PCNA, POLQ, RPA3, TOP2A, and UBE2V2) was associated with poor prognosis, and the expression of one gene (ATM) was associated with good prognosis. Furthermore, the prognostic value of specific genes did not correlate with proliferation. A few genes were associated with chemotherapy response in BC subtypes and treatment-specific manner. In ER-negative/HER2-negative tumors, the MSH2, MSH6, and FAN1 (previously MTMR15) genes were associated with pathological complete response and residual invasive cancer in taxane/anthracycline-treated patients. Conversely, PMS2 expression was associated with residual invasive cancer in treatments using anthracycline as a single agent. In HER2-positive tumors, TOP2A was associated with patient response to anthracyclines but not to taxane/anthracycline regimens. In genes expressed in a bimodal fashion, RECQL4 was significantly associated with clinical outcome. In vitro studies showed that defects in RECQL4 impair homologous recombination, sensitizing BC cells to DNA-damaging agents.

Background. This randomized phase II trial was designed to compare the rate of pathologic complete response (pCR) induced by neoadjuvant cyclophosphamide plus doxorubicin (AC) followed by ixabepilone or paclitaxel in women with early stage breast cancer (BC). Expression of βIII-tubulin as a predictive marker was also evaluated. Patients and Methods. Women with untreated, histologically confirmed primary invasive breast adenocarcinoma received four cycles of AC followed by 1:1 randomization to either ixabepilone 40 mg/m2 (3-hour infusion) every 3 weeks for four cycles (n = 148) or weekly paclitaxel 80 mg/m2 (1-hour infusion) for 12 weeks (n = 147). All patients underwent a core needle biopsy of the primary cancer for molecular marker analysis prior to chemotherapy. βIII-Tubulin expression was assessed using immunohistochemistry. Results. There was no significant difference in the rate of pCR in the ixabepilone treatment arm (24.3%; 90% confidence interval [CI], 18.6–30.8) and the paclitaxel treatment arm (25.2%; 90% CI, 19.4–31.7). βIII-Tubulin-positive patients obtained higher pCR rates compared with βIII-tubulin-negative patients in both treatment arms; however, βIII-tubulin expression was not significantly associated with a differential response to ixabepilone or paclitaxel. The safety profiles of both regimens were generally similar, although neutropenia occurred more frequently in the ixabepilone arm (grade 3/4: 41.3% vs. 8.4%). The most common nonhematologic toxicity was peripheral neuropathy. Conclusions. Neoadjuvant treatment of early stage BC with AC followed by ixabepilone every 3 weeks or weekly paclitaxel was well tolerated with no significant difference in efficacy. Higher response rates were observed among βIII-tubulin-positive patients.

Purpose: Patients with triple-negative breast cancer (TNBC) and residual disease after neoadjuvant chemotherapy generally have worse outcome; however, some patients with residual tumor after neoadjuvant chemotherapy do not relapse. We hypothesize that there are subgroups of patients with chemoresistant TNBC with different prognosis. Experimental Design: Forty-nine chemoresistant cases from 111 patients with TNBC treated with neoadjuvant chemotherapy (M.D. Anderson Cancer Center, Houston, TX) constituted the discovery cohort, and 25 chemoresistant samples from 47 neoadjuvant chemotherapy-treated TNBC (The Methodist Hospital, Houston, TX) were chosen for validation. Extended validation was carried out in 269 operable TNBC predicted to be chemoresistant by expression pattern from published datasets. Results: We established a seven-gene prognostic signature using dChip and gene set enrichment analyses. In the independent validation cohort, the classifier predicted correctly with positive predictive value of 75.0% and negative predictive value (i.e., relapse-free survival; RFS) of 76.9% at 3 years. Those predicted to relapse had a HR of 4.67 [95% confidence interval (CI): 1.27–17.15] for relapse in 3 years. In extended validation, patients predicted not to relapse exhibited 3-year RFS of 78.9%, whereas the 3-year RFS was 48.5% for patients predicted to relapse, with HR of 2.61 (95% CI: 1.52–4.49). The TNBC subgroup that predicted to have relatively favorable prognosis was characterized by high expression of “luminal-like” genes [androgen-receptor (AR) and GATA3], whereas the subgroup with worse prognosis was characterized by expression of cancer stem-cell markers. Conclusion: We developed a clinically relevant signature for patients with chemoresistant TNBC. For these women, new therapeutic strategies like targeting AR activation or cancer stem cells may need to be developed. Clin Cancer Res; 19(10); 2723–33. ©2013 AACR.

BackgroundThe heterogeneity of breast cancer makes identifying effective therapies challenging. The I-SPY 2 trial, a multicenter, adaptive phase 2 trial of neoadjuvant therapy for high-risk clinical stage II or III breast cancer, evaluated multiple new agents added to standard chemotherapy to assess the effects on rates of pathological complete response (i.e., absence of residual cancer in the breast or lymph nodes at the time of surgery). MethodsWe used adaptive randomization to compare standard neoadjuvant chemotherapy plus the tyrosine kinase inhibitor neratinib with control. Eligible women were categorized according to eight biomarker subtypes on the basis of human epidermal growth factor receptor 2 (HER2) status, hormone-receptor status, and risk according to a 70-gene profile. Neratinib was evaluated against control with regard to 10 biomarker signatures (prospectively defined combinations of subtypes). The primary end point was pathological complete response. Volume changes on serial magnetic reson...

The use of neoadjuvant systemic therapy for the treatment of breast cancer patients is increasing. Pathologic response in the form of pathologic complete response (pCR) and grading systems of partial response, such as the residual cancer burden (RCB) system, gives valuable prognostic information for patients and is used as a primary endpoint in clinical trials. The breast cancer and pathology communities are responding with efforts to standardize pathology in patients receiving neoadjuvant chemotherapy. In this review, we summarize the challenges that postneoadjuvant systemic therapy surgical specimens pose and how pathologists and the multidisciplinary team can work together to optimize handling of these specimens. Multidisciplinary communication is essential. A single, standardized approach to macroscopic and microscopic pathologic examination makes it possible to provide reliable response information. This approach employs a map of tissue sections to correlate clinical, gross, microscopic, and imaging findings in order to report the presence of pCR (ypT0 ypN0 and ypT0/is ypN0) versus residual disease, the ypT and ypN stage using the current AJCC/UICC staging system, and the RCB.

Importance We previously described and validated a breast cancer staging system (CPS+EG, clinical-pathologic scoring system incorporating estrogen receptor–negative disease and nuclear grade 3 tumor pathology) for assessing prognosis after neoadjuvant chemotherapy using pretreatment clinical stage, posttreatment pathologic stage, estrogen receptor (ER) status, and grade. Development of the CPS+EG staging system predated routine administration of trastuzumab in patients with ERBB2 -positive disease (formerly HER2 or HER2/neu ). Objective To validate the CPS+EG staging system using the new definition of ER positivity (≥1%) and to develop an updated staging system (Neo-Bioscore) that incorporates ERBB2 status into the previously developed CPS+EG. Design, Setting, and Participants Retrospective review of data collected prospectively from January 2005 through December 2012 on patients with breast cancer treated with neoadjuvant chemotherapy at The University of Texas MD Anderson Cancer Center. Main Outcomes and Measure Prognostic scores were computed using 2 versions of the CPS+EG staging system, one with ER considered positive if it measured 10% or higher, the other with ER considered positive if it measured 1% or higher. Fits of the Cox proportional hazards model for the 2 sets of prognostic scores were compared using the Akaike Information Criterion (AIC). Status of ERBB2 was added to the model, and the likelihood ratio test was used to determine improvement in fit. Results A total of 2377 patients were included; all were women (median age, 50 years [range, 21-87 years]); ER status was less than 1% in 28.9%, 1% to 9% in 8.3%, and 10% or higher in 62.8%; 591 patients were ERBB2 positive. Median follow-up was 4.2 years (range, 0.5-11.7 years). Five-year disease-specific survival was 89% (95% CI, 87%-90%). Using 1% or higher as the cutoff for ER positivity, 5-year disease-specific survival estimates determined using the CPS+EG stage ranged from 52% to 98%, thereby validating our previous finding that the CPS+EG score facilitates more refined categorization into prognostic subgroups than clinical or final pathologic stage alone. The AIC value for this model was 3333.06, while for a model using 10% or higher as the cutoff for ER positivity, it was 3333.38, indicating that the model fits were nearly identical. The improvement in fit of the model when ERBB2 status was added was highly significant, with 5-year disease-specific survival estimates ranging from 48% to 99% ( P ERBB2 into the staging system defined the Neo-Bioscore, which provided improved stratification of patients with respect to prognosis. Conclusions and Relevance The Neo-Bioscore improves our previously validated staging system and allows its application in ERBB2 -positive patients. We recommend that treatment response and biologic markers be incorporated into the American Joint Committee on Cancer staging system.

To successfully translate genomic classifiers to the clinical practice, it is essential to obtain reliable and reproducible measurement of the classifier performance. A point estimate of the classifier performance has to be accompanied with a measure of its uncertainty. In general, this uncertainty arises from both the finite size of the training set and the finite size of the testing set. The training variability is a measure of classifier stability and is particularly important when the training sample size is small. Methods have been developed for estimating such variability for the performance metric AUC (area under the ROC curve) under two paradigms: a smoothed cross-validation paradigm and an independent validation paradigm. The methodology is demonstrated on three clinical microarray datasets in the microarray quality control consortium phase two project (MAQC-II): breast cancer, multiple myeloma, and neuroblastoma. The results show that the classifier performance is associated with large variability and the estimated performance may change dramatically on different datasets. Moreover, the training variability is found to be of the same order as the testing variability for the datasets and models considered. In conclusion, the feasibility of quantifying both training and testing variability of classifier performance is demonstrated on finite real-world datasets. The large variability of the performance estimates shows that patient sample size is still the bottleneck of the microarray problem and the training variability is not negligible.

The use of neoadjuvant systemic therapy (NST) for the treatment of primary breast cancer has constantly increased, especially in trials of new therapeutic regimens. In the 1980 s, NST was shown to substantially improve breast-conserving surgery rates and was first typically used for patients with inoperable locally advanced or inflammatory breast cancer. Investigators have since also used NST as an in vivo test for chemosensitivity by assessing pathologic complete response. Today, by using pathologic response and other biomarkers as intermediate end points, results from trials of new regimens and therapies that use NST are aimed to precede and anticipate the results from larger adjuvant trials. In 2003, a panel of representatives from various breast cancer clinical research groups was first convened in Biedenkopf to formulate recommendations on the use of NST. The obtained consensus was updated in two subsequent meetings in 2004 and 2006. The most recent conference on recommendations on the use of NST took place in 2010 and forms the basis of this report.

Reliable stability and yield of RNA from breast cancer tissues are important for biobanking, clinical trials, and diagnostic testing.Aliquots of fresh primary tumor tissue from 17 surgically resected invasive breast cancers were placed into RNAlater at room temperature after tumor removal (baseline) and up to 3 hours thereafter or were snap frozen at baseline and 40 minutes thereafter. Samples were stored at -80°C until gene expression profiling with Affymetrix Human Gene U133A microarrays. We evaluated the effects of cold ischemic time (the time from tumor specimen removal to sample preservation) and sample preservation method on RNA yield, Bioanalyzer-based RNA integrity number, microarray-based 3'/5' expression ratios for assessing transcript integrity, and microarray-based measurement of single-gene and multigene expression signatures. The statistical significance of the effects was assessed using linear mixed effects regression models. All statistical tests were two-sided.Sample preservation in RNAlater statistically significantly improved RNA integrity compared with snap freezing as assessed by the RNA integrity number, which increased from 7.31 to 8.13 units (difference = 0.82 units, 95% confidence interval [CI] = 0.53 to 1.11 units, P.001), and RNA yield, which increased threefold from 8.9 to 28.6 μg (difference = 19.7 μg, 95% CI = 14.1 to 25.3 μg, P.001). Prolonged cold ischemic delay at room temperature before sample stabilization decreased the RNA integrity number by 0.12 units/h (95% CI = 0.02 to 0.23 units/h) compared with a projected average RNA integrity number of 7.39 if no delays were present (P = .008) and decreased the RNA yield by 1.5 μg/h (95% CI = 0 to 4 μg/h) from a baseline mean RNA yield of 33.5 μg if no delays were present (P = .019). Prolonged cold ischemia statistically significantly increased the 3'/5' ratio of control gene transcripts, particularly of STAT1 (P.001). Snap freezing statistically significantly increased the 3'/5' ratio of three control transcripts (ACTB, GAPDH, and 18S rRNA). Expression levels of single genes and multigene signatures for breast cancer were largely unaffected by sample preservation method or cold ischemia.Sample preservation in RNAlater improves RNA yield and quality, whereas cold ischemia increases RNA fragmentation as measured by the 3'/5' expression ratio of control genes. However, expression levels of single genes and multigene signatures that are of diagnostic relevance in breast cancer were mostly unaffected by sample preservation method or prolonged cold ischemic duration.

Purpose: Estrogen receptor-positive (ER+) and -negative (ER) breast cancers are molecularly distinct diseases. We hypothesized that p53 mutations may lead to different transcriptional changes and carry different prognostic value in these two different types of cancers. Experimental Design: We developed a 39-gene p53 signature derived from 213 ER+ and a separate 30-gene signature from 38 ER− cancers with known mutation status and tested their prognostic and chemotherapy response predictive values in ER+ and ER− cancers, respectively. Results: External validation to predict p53 status (n = 103) showed sensitivity and specificity of 89% and 54% for the 39-gene signature, and 82% and 61% for the 30-gene signature. The 39-gene signature was predictive of worse distant metastasis free survival in ER+ cancers in two separate prognostic data sets (n = 255, HR: 2.3, P = 0.005 and n = 198, HR: 2.17, P = 0.09). It also predicted for poor prognosis even with adjuvant tamoxifen therapy (n = 277, HR = 2.43, P < 0.0001) but it was not prognostic in ER− cancers. It was also associated with higher chemotherapy sensitivity in ER+ but not in ER− cancers. The prognostic and predictive values remained significant in multivariate analysis. The 30-gene, ER−, p53 signature showed no prognostic or predictive values in ER+ cancers but it was associated with better prognosis in ER− cancers. It also had no chemotherapy response predictive value in ER− or ER+ cancers. Conclusions: P53 dysfunction is prognostically most relevant in ER+ cancers and supports the hypothesis that different predictive or prognostic markers will be needed for different molecular subsets of breast cancer. Clin Cancer Res; 17(8); 2591–601. ©2011 AACR.

The goal of this study was to determine whether gene expression differences exist between inflammatory breast cancers (IBC) and T stage-matched non-IBC patients stratified by hormone receptor and HER2 status. We used Affymetrix GeneChips to analyze 82 tumor samples (25 T4d patients, and 57 T4a-c patients) of newly diagnosed breast cancers. Genes that were differentially expressed between the IBC and non-IBC specimens were identified using the t test, and differential expression of gene sets was assessed using gene set analysis. Three distinct clinical subtypes of IBC and non-IBC were compared: ER-positive/HER2-normal, HER2-amplified, and ER-negative/HER2-normal. When we compared expression data from all IBC with all non-IBC, we found no significant differences after adjusting for multiple testing. When IBC and non-IBC tumors were compared by clinical subtype, however, significant differences emerged. Complement and immune system-related pathways were overexpressed in ER-positive/HER2-normal IBC. Protein translation and mTOR signaling were overexpressed in HER2-amplified IBC. Apoptosis-, neural-, and lipid metabolism-related pathways were overexpressed in ER-negative/HER2-normal IBC compared with non-IBC of the same receptor phenotype. In this T stage-matched case-control study, the survival curves of patients with IBC and non-IBC were similar for all three clinical subtypes. IBC tumors can be divided into molecular and clinical subtypes similar to those of non-IBC. Clinical subtypes of IBC show molecular differences compared with similar subtypes of non-IBC.

BACKGROUND: Although the use of SSM is becoming more common, there are few data on long-term, local-regional, and distant recurrence rates after treatment. The purpose of this study was to examine the rates of local, regional, and systemic recurrence, and survival in breast cancer patients who underwent skin-sparing mastectomy (SSM) or conventional mastectomy (CM) at our institution. METHODS: Patients with stage 0 to III unilateral breast cancer who underwent total mastectomy at our center from 2000 to 2005 were included in this study. Kaplan-Meier curves were calculated, and the log-rank test was used to evaluate the differences between overall and disease-free survival rates in the 2 groups. RESULTS: Of 1810 patients, 799 (44.1%) underwent SSM and 1011 (55.9%) underwent CM. Patients who underwent CM were older (58.3 vs 49.3 years, P

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.

Purpose Although most breast cancer patients who receive neoadjuvant chemotherapy (NCT) have a tumor response, a small proportion experience progressive disease (PD). Predictors of response have been reported, but predictors for progression have not been identified. We sought to identify predictors of tumor progression during NCT with the ultimate aim of identifying patients who might benefit from a first-line surgical approach or from novel targeted therapies. Patients and Methods Data were obtained from reviewing medical records of patients with stage I to III breast cancer who received NCT (anthracycline and/or taxane based). Statistical analysis was performed to compare patients with any response or stable disease with patients with PD. Results One thousand nine hundred twenty-eight patients received NCT; 1,762 patients (91%) had some response, 107 (6%) had stable disease, and 59 (3%) had PD at some point during NCT. Factors predictive of PD included African American race (P = .002), tumor (T) status (P = .002), and American Joint Committee on Cancer clinical stage (P = .02). Histopathologic features of PD were high tumor grade (P = .005), high Ki-67 score (P = .002), and negative estrogen receptor (ER)/progesterone receptor (PR) status (P < .001/P < .001). Pre-NCT T status, race, and ER status were independent predictors of progression in multivariate analysis. Disease progression was a negative predictor of distant disease–free survival and overall survival in multivariate analysis (P < .001). Conclusion Factors predictive of PD include race, advanced tumor stage, high nuclear grade, high Ki-67 score, and ER/PR negativity. Because many of these variables are also associated with response to NCT, novel molecular predictors are needed to identify patients at risk for progression on standard NCT.

This study specifically addressed the performance of chromogenic in situ hybridization (CISH) on HER2 testing in 66 breast carcinomas with chromosome 17 polysomy and 49 carcinomas with an equivocal HercepTest (DakoCytomation, Carpinteria, CA) score by comparing CISH with corresponding FISH results at 2 test sites and evaluating intersite agreement of CISH results. For tumors with chromosome 17 polysomy, when using the manufacturers’ criteria, the concordance values between CISH and FISH at site A, site B, and intersite CISH agreement were 95.8%, 95.5%, and 93.5%, respectively; when using the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) criteria, the values were 100.0%, 100.0%, and 100.0%, respectively. For tumors with an equivocal HercepTest score, when using the manufacturers’ criteria, the concordance values between the 2 methods at site A, site B, and intersite CISH agreement were 88.2%, 95.1%, and 91.1%, respectively; when using the ASCO/CAP criteria, the values were 96.7%, 97.3%, and 97.4%, respectively. These results indicate that CISH is reliable for testing these 2 types of tumors, especially when the ASCO/CAP criteria are used.

Chromogenic in situ hybridization (CISH) has shown the potential to replace fluorescence in situ hybridization (FISH) to determine HER2 gene status. To validate the reliability of CISH, we used 226 consecutive breast carcinomas from 2 institutions and tested CISH and FISH on the same tumor set simultaneously at different test sites. Besides manufacturers' scoring criteria, the new American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines were used to interpret HER2 status. The concordance between CISH and FISH for positive and negative results was 98.5% at site A and 98.6% at site B using the manufacturers' criteria, and 99.0% at site A and 99.1% at site B using the ASCO/CAP criteria. Reproducibility of CISH results was more than 98.0% among 3 sites using the manufacturers' criteria and 100.0% between 2 sites using the ASCO/CAP criteria. Our results confirm that CISH is reliable for HER2 testing per ASCO/CAP guidelines.

The human epidermal growth factor receptor (HER-2) oncogene encodes a transmembrane tyrosine kinase receptor that has evolved as a major classifier of invasive breast cancer and target of therapy for the disease. The validation of the general prognostic significance of HER-2 gene amplification and protein overexpression in the absence of anti-HER-2 targeted therapy is discussed in a study of 107 published studies involving 39,730 patients, which produced an overall HER-2-positive rate of 22.2% and a mean relative risk for overall survival (OS) of 2.74. The issue of HER-2 status in primary versus metastatic breast cancer is considered along with a section on the features of metastatic HER-2-positive disease. The major marketed slide-based HER-2 testing approaches, immunohistochemistry, fluorescence in situ hybridization, and chromogenic in situ hybridization, are presented and contrasted in detail against the background of the published American Society of Clinical Oncology-College of American Pathologists guidelines for HER-2 testing. Testing issues, such as the impact of chromosome 17 polysomy and local versus central HER-2 testing, are also discussed. Emerging novel HER-2 testing techniques, including mRNA-based testing by real-time polymerase chain reaction and DNA microarray methods, HER-2 receptor dimerization, phosphorylated HER-2 receptors, and HER-2 status in circulating tumor cells, are also considered. A series of biomarkers potentially associated with resistance to trastuzumab is discussed with emphasis on the phosphatase and tensin homologue deleted on chromosome ten/Akt and insulin-like growth factor receptor pathways. The efficacy results for the more recently approved small molecule HER-1/HER-2 kinase inhibitor lapatinib are also presented along with a more limited review of markers of resistance for this agent. Additional topics in this section include combinations of both anti-HER-2 targeted therapies together as well as with novel agents including bevacizumab, everolimus, and tenespimycin. A series of novel HER-2-targeting agents is also presented, including pertuzumab, ertumaxomab, HER-2 vaccines, and recently discovered tyrosine kinase inhibitors. Biomarkers predictive of HER-2 targeted therapy toxicity are included, and the review concludes with a consideration of HER-2 status in the prediction of response to non-HER-2 targeted treatments including hormonal therapy, anthracyclines, and taxanes.

Purpose: We used high-resolution oligonucleotide comparative genomic hybridization (CGH) arrays and matching gene expression array data to identify dysregulated genes and to classify breast cancers according to gene copy number anomalies. Experimental Design: DNA was extracted from 106 pretreatment fine needle aspirations of stage II-III breast cancers that received preoperative chemotherapy. CGH was done using Agilent Human 4 × 44K arrays. Gene expression data generated with Affymetrix U133A gene chips was also available on 103 patients. All P values were adjusted for multiple comparisons. Results: The average number of copy number abnormalities in individual tumors was 76 (range 1-318). Eleven and 37 distinct minimal common regions were gained or lost in >20% of samples, respectively. Several potential therapeutic targets were identified, including FGFR1 that showed high-level amplification in 10% of cases. Close correlation between DNA copy number and mRNA expression levels was detected. Nonnegative matrix factorization (NMF) clustering of DNA copy number aberrations revealed three distinct molecular classes in this data set. NMF class I was characterized by a high rate of triple-negative cancers (64%) and gains of 6p21. VEGFA, E2F3, and NOTCH4 were also gained in 29% to 34% of triple-negative tumors. A gain of ERBB2 gene was observed in 52% of NMF class II and class III was characterized by a high rate of estrogen receptor–positive tumors (73%) and a low rate of pathologic complete response to preoperative chemotherapy (3%). Conclusion: The present study identified dysregulated genes that could classify breast cancer and may represent novel therapeutic targets for molecular subsets of cancers.

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy–based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor–positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor–positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor–positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor–positive breast cancer. [Cancer Res 2008;68(15):6084–91]

Learning Objectives After completing this course, the reader will be able to: Define survival outcomes in women with early-stage breast cancer who achieve pathological complete response following primary systemic chemotherapy.Define the prognostic value of initial clinical stage in women with breast cancer who achieve pathological complete response following primary systemic chemotherapy.Define survival outcomes in women with inflammatory breast cancer who achieve pathological complete response following primary systemic chemotherapy. CME Access and take the CME test online and receive 1 AMA PRA Category 1 Credit™ at CME.TheOncologist.com The aim of this retrospective study was to determine the prognostic impact of initial clinical stage in patients who achieved a pathological complete response (pCR) after receiving primary systemic chemotherapy (PST). Between 1977 and 2006, 489 patients who had achieved a pCR after receiving an anthracycline-based PST regimen were identified. Recurrence-free survival (RFS) and overall survival (OS) were estimated with the Kaplan–Meier product limit method and the differences between groups were compared using the log-rank statistic. Cox proportional hazards models were fit to determine the association of initial clinical stage with survival outcomes after adjusting for patient and tumor characteristics. The median age was 47 years. Twenty (4.1%) patients had stage I disease, 243 (49.7%) had stage II disease, 189 (38.7%) had stage III disease, and 37 (7.5%) had inflammatory breast cancer (IBC). At a median follow-up of 45 months, 59 (12%) patients had experienced disease recurrence. The 5-year RFS and OS rates for the whole cohort were 87.8% and 89.3%, respectively. Lower clinical stage at diagnosis was associated with statistically significant higher RFS and OS rates. In a multivariate model, patients with clinical stage IIIB/C disease and those with IBC had lower RFS rates than patients with clinical stage I/II/IIIA disease. In addition, patients with clinical stage IIIB/C disease and those with IBC had a greater hazard of death than patients with clinical stage I/II/IIIA disease. Overall, patients who achieved a pCR had a low rate of recurrence. However, higher clinical stage and IBC were associated with worse outcomes in breast cancer patients who achieved a pCR after PST.

Triple negative breast carcinomas (TNBCs) are a group of primary breast tumors with aggressive clinical behavior. Most TNBCs possess a basal phenotype (BP) and show varying degrees of basal cytokeratin and myoepithelial marker expression. The importance of recognizing these tumors came to light largely as the result of gene expression profiling studies that categorized breast cancer into 3 major groups. Two of these groups are defined by their respective expression of estrogen receptor and HER2. TNBCs represent a third group and are defined by negativity for hormone receptors and HER2. TNBCs currently lack effective targeted therapies and are frequently resistant to standard chemotherapeutic regimens. These tumors tend to occur in premenopausal women and members of specific ethnic groups and a subset are associated with heritable BRCA1 mutations. For patients with sporadic TNBCs and BP tumors, BRCA1 dysfunction seems to play a major role in the development and progression of disease. The pathologist's role in the diagnosis and characterization of TNBCs and BP tumors is currently being defined as we are acquiring knowledge of the biologic and genetic underpinnings that drive this heterogeneous group of diseases. This review will provide a historical prospective on TNBCs and tumors that express basal cytokeratins and myoepithelial makers. Additionally, we will discuss the molecular biologic, genetic and pathologic aspects of these tumors. Guidelines will be provided on how to best approach the diagnosis of these cases and on what input pathologists should provide clinicians to help develop optimal therapeutic and preventative strategies against this aggressive group of breast cancers.

Background Mucinous, medullary, and tubular carcinomas are uncommon types of breast cancer whose rarity does not permit large single-institution studies or randomized trials to define optimal treatments. In this study, we evaluated the long-term outcomes of breast-conserving therapy (BCT) for these subtypes of breast cancer and compared them with those for invasive ductal carcinoma. Methods In our institutional database of patients who received BCT from 1965 to 1999, 1,643 patients with stage I to II mucinous (61), medullary (37), tubular (60), and invasive ductal (1,485) histologies were identified. The clinical and pathologic features of the 4 groups were evaluated and compared with respect to local-regional recurrence rates, disease-free survival, and overall survival (OS). Results No statistically significant differences were found in the local-regional failure rate among the 4 groups (10.6-year median follow-up). Only patients with tubular carcinoma had better 5- and 10-year OS rates ( P = .013). In multivariable analysis, factors associated with improved OS included age at or below 50 years, negative nodal status, use of chemotherapy or hormonal therapy, and tubular histology. Conclusions BCT for mucinous, medullary, or tubular carcinoma resulted in similar local-regional failure rates to that for invasive ductal carcinoma. Tubular carcinoma patients had the most favorable OS. BCT is an appropriate treatment strategy for early-stage mucinous, medullary, and tubular carcinomas.

Purpose: We examined whether the response predicted by a 30-gene pharmacogenomic test correlated with the residual cancer burden (RCB) after preoperative chemotherapy with paclitaxel, 5-fluorouracil, doxorubicin, and cyclophosphamide (T/FAC). Experimental Design: Gene expression profiling was done at diagnosis in 74 patients with stages I to III breast cancer and was used to calculate a pharmacogenomic score and predict response to chemotherapy [pathologic complete response (pCR) or residual disease (RD)]. All patients received 6 months of preoperative T/FAC. Following pathologic review, a RCB score was calculated based on residual tumor and lymph node features. Four RCB classes were assigned; RCB-0 (pCR), RCB-I (near-PCR), RCB-II (moderate RD), and RCB-III (extensive RD). The correlations between the pharmacogenomic score, predicted pathologic response, RCB score, and RCB class were examined. Results: Thirty-three patients were predicted to have pCR, and 40 were predicted to have RD. Observed responses were RCB-0: n = 20 (27%); RCB-I: n = 5 (7%); RCB-II: n = 36 (49%); and RCB-III: n = 13 (16%) patients. Pharmacogenomic and RCB scores were correlated (Pearson's R = −0.501, P < 0.0001). There was no difference between the mean genomic predictor scores for RCB-0/I groups (P = 0.94), but these were different from the mean scores of the RCB-II/III groups (P < 0.001). Among the 25 patients with RCB-0/I response, 19 (76%) were predicted to achieve pCR. The pharmacogenomic test correctly predicted RD in 92% of the patients with RCB-III, which corresponds to chemotherapy-resistant disease. Conclusions: The 30-gene pharmacogenomic test showed good correlation with the extent of residual invasive cancer burden measured as both continuous and categorical variables.

We examined the correlation between HER2 expression and pathologic complete response (pCR) to paclitaxel/FAC (T/FAC) preoperative chemotherapy in breast cancer. Retrospective analysis of data including 534 patients treated with preoperative T/FAC was performed. Gene expression results were available from two datasets of 132 and 286 patients, and were used to examine the co-expression of HER2 and topoisomerase II α (TOP2A) and microtubule associated protein tau (MAP-Tau). Of the 534 patients, 105 (20%) had HER2-overexpressing breast cancer. The pCR rates were 33% and 15% for patients with HER2+ and HER2- tumors (P