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Context.— Neoadjuvant systemic therapy refers to the use of systemic agent(s) for malignancy prior to surgical treatment and has recently emerged as an option for most breast cancer patients eligible for adjuvant systemic therapy. Consequently, treated breast carcinomas have become routine specimens in pathology practices. A standard protocol has not yet been universally adopted for the evaluation and reporting of these specimens. The American Joint Committee on Cancer staging system recognizes the challenges in staging breast carcinomas after neoadjuvant treatment and provides important data points but does not currently provide detailed guidance in estimating the residual tumor burden in the breast and lymph nodes. The Residual Cancer Burden system is the only Web-based system that quantifies treatment response as a continuous variable using residual tumor burden in the breast and the lymph nodes. Objective.— To provide clarifications and guidance for evaluation and reporting of postneoadjuvant breast specimens, discuss issues with the current staging and reporting systems, and provide specific suggestions for future modifications to the American Joint Committee on Cancer system and the Residual Cancer Burden calculator. Data Sources.— English-language literature on the subject and the data from the I-SPY 2, a multicenter, adaptive randomization phase 2 neoadjuvant platform trial for early-stage, high-risk breast cancer patients. Conclusions.— This article highlights challenges in the pathologic evaluation and reporting of treated breast carcinomas and provides recommendations and clarifications for pathologists and clinicians. It also provides specific recommendations for staging and discusses future directions.

BACKGROUND We proposed that a test for sensitivity to the adjuvant endocrine therapy component of treatment for patients with stage II-III breast cancer (SET2,3) should measure transcription related to estrogen and progesterone receptors (SETER/PR index) adjusted for a baseline prognostic index (BPI) combining clinical tumor and nodal stage with molecular subtype by RNA4 (ESR1, PGR, ERBB2, and AURKA). PATIENTS AND METHODS Patients with clinically high-risk, hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative (HR+/HER2-) breast cancer received neoadjuvant taxane-anthracycline chemotherapy, surgery with measurement of residual cancer burden (RCB), and then adjuvant endocrine therapy. SET2,3 was measured from pre-treatment tumor biopsies, evaluated first in an MD Anderson Cancer Center (MDACC) cohort (n = 307, 11 years' follow-up, U133A microarrays), cut point was determined, and then independent, blinded evaluation was carried out in the I-SPY2 trial (n = 268, high-risk MammaPrint result, 3.8 years' follow-up, Agilent-44K microarrays, NCI Clinical Trials ID: NCT01042379). Primary outcome measure was distant relapse-free survival. Multivariate Cox regression models tested prognostic independence of SET2,3 relative to RCB and other molecular prognostic signatures, and whether other prognostic signatures could substitute for SETER/PR or RNA4 components of SET2,3. RESULTS SET2,3 added independent prognostic information to RCB in the MDACC cohort: SET2,3 [hazard ratio (HR) 0.23, P = 0.004] and RCB (HR 1.77, P < 0.001); and the I-SPY2 trial: SET2,3 (HR 0.27, P = 0.031) and RCB (HR 1.68, P = 0.008). SET2,3 provided similar prognostic information irrespective of whether RCB-II or RCB-III after chemotherapy, and in both luminal subtypes. Conversely, RCB was most strongly prognostic in cancers with low SET2,3 status (MDACC P < 0.001, I-SPY2 P < 0.001). Other molecular signatures were not independently prognostic; they could effectively substitute for RNA4 subtype within the BPI component of SET2,3, but they could not effectively substitute for SETER/PR index. CONCLUSIONS SET2,3 added independent prognostic information to chemotherapy response (RCB) and baseline prognostic score or subtype. Approximately 40% of patients with clinically high-risk HR+/HER2- disease had high SET2,3 and could be considered for clinical trials of neoadjuvant endocrine-based treatment.

Background: I-SPY 2 is a neoadjuvant platform trial open to patients with locally advanced, molecular high-risk breast cancer. In a concerted pursuit of mid-therapy response biomarkers, we evaluated inter-regimen biopsies, to identify patients who may be candidates for treatment de-escalation. In a pilot study, we observed that absence of carcinoma in an inter-regimen biopsy may predict pathologic complete response (pCR). In this expanded study of 100 participants, we sought to confirm that finding and assess pathologic features of the inter-regimen biopsy as predictors of tumor response to neoadjuvant therapy. Methods: Digital H&E images of 100 inter-regimen (12 week) image-guided breast biopsies +/- ancillary immunohistochemistry (p63 and/or cytokeratin) were reviewed by 9 I-SPY affiliated pathologists to record 1) tumor bed and 2) presence/absence of residual invasive carcinoma (IC) (with tumor cellularity scored as 0-100%). The data set included 393 cores (mean 3.9 (2-4) cores/biopsy). Fisher’s exact t-test was used for association of presence/absence of IC with pCR, and tumoral hormone receptor (HR) and HER2 status. Association between biopsy tumor cellularity and residual cancer burden (RCB) indices used Pearson’s correlation. Results: In the biopsy set, 84 (84%) had ≥80% inter-observer diagnostic agreement on both 1) presence of tumor bed and 2) presence/absence of IC (53 IC+ /31 IC-). IC+/IC- biopsies had equal numbers of evaluable tissue cores. The primary tumors were 63% HR+/37% HR-. The presence of IC in the biopsy correlated with tumoral HR/HER2 status (p=0.0014: 74%: HR+HER2-; 62%: TN; 60%: HR+HER2+; 10%: HR-HER2+). Of 31 patients with IC- biopsies, 25 (80%) went on to pCR, whereas only 7/53 (13%) of patients with IC+ biopsies had pCR, conferring an odds ratio for pCR of 26, Fisher p=7.5E-10. Overall, IC- biopsies had a positive predictive value (PPV) for pCR of 81%, with a PPV for HR- tumors of 94% vs. 67% for HR+ tumors (Table 1). In the 6 IC- biopsies from patients with non-pCR (“false-negatives”), most were HR+ (5/6, Table 1), and tumor bed size in the resection specimen was smaller than for IC+ biopsies with non-pCR: 276 mm2 (0.4-1000 mm2) vs. 1166 mm2 (1-11960 mm2). In contrast, the 46/53 IC+ biopsies in patients with non-pCR had a PPV for predicting non-pCR of 86%, (PPV for HR+ tumors: 94% vs. PPV for HR- tumors: 66%. Tumor cellularity in the biopsy (mean 37%, [2.5-93%]) did not correlate with RCB index (p=0.57) or RCB breast-only index (p = 0.17) at resection. Conclusion: In this 100 biopsy set from the I-SPY2 trial, the absence of residual carcinoma in inter-regimen biopsies was highly predictive of pCR, particularly for HR- tumors. The “false-negative” biopsies (IC-/non-pCR) were predominantly HR+ tumors with small residual tumor beds at resection. Conversely, the presence of carcinoma in inter-regimen biopsies was highly predictive of non-pCR, particularly for HR+ tumors. These data demonstrate the utility, and the limitations, of the inter-regimen biopsy as one tool to identify patients who may benefit from therapeutic de-escalation. Table 1: PPV for pCR/non-PCR by Inter-regimen Biopsy StatusInter-regimen biopsy with or without Invasive carcinoma (IC+/-)pCRnon-pCRPPV (Sensitivity) for pCR(IC- Biopsies)PPV (Sensitivity) for non-pCR(IC+ biopsies)IC- biopsiesAll25681% (78%)-HR+10567% (83%)-HR-15194% (75%)-IC+ biopsiesAll746-86% (88%)HR+236-94% (88%)HR-510-66% (91%) Citation Format: Jodi M Carter, Molly E Klein, Sara J Venters, Kimmie Rabe, I Tolgay Ocal, Kamaljeet Singh, Denise M Wolf, Sunati Sahoo, Shuko Harada, Laila Khazai, Malini Harigopal, Alexander D Borowsky, Gregor Krings, Ronald Balassanian, Yunn-Yi Chen, Kimberley Cole, Sonal Shad, Amy Delson, Lamorna Brown-Swigart, I-SPY 2 TRIAL Consortium, Laura Esserman, Laura van ‘t Veer, W Fraser Symmans. Pathologic features of the inter-regimen biopsy predict response to neoadjuvant therapy in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-09.

Background: The I-SPY 2 TRIAL, open to patients with locally advanced, molecular high-risk breast cancer, aims to bring each patient to pathologic complete response (pCR) with a minimum of toxicity. Here we test the hypothesis that imaging (MR volume predictors) combined with core biopsy may be used to accurately select candidates who show early response and provide an option of treatment de-escalation at mid-therapy (12 weeks). Methods: Of 100 I-SPY 2 patients with pathologist-assessed core biopsies at the inter-regimen time point (~12 weeks through treatment) and pCR data, 87 also had serial MR images and were considered in this study. Eleven I-SPY 2 TRIAL pathologists independently provided a digital assessment of the presence or absence of residual invasive cancer from H&E stained, and any requested ancillary IHC, images from imaging-guided core biopsies. Pathology predicts pCR if there is a consensus of no invasive residual disease. We generated predictions for all (55) unique pairs over the 11 pathologists, where pCR is predicted if both pathologists find no invasive cells. MRI pCR prediction models were previously developed on an independent dataset of ~990 I-SPY 2 patients, and applied to this cohort. Volume-based prediction models were previously optimized within each subtype and predicted probability thresholds were selected over a range of positive predictive value (PPV). In this study, MR predicts pCR (positive test) if the predicted probability is above a threshold that yields a given PPV value. For each pathologist pair, we combined pathology-based and MR-based predictors into a predictive-RCB (pre-RCB); and pre-RCB predicts a patient as pCR (RCB0) if both MR and pathology predicts pCR. Predictive performance is assessed by calculating the mean and range of PPV and sensitivity.Results: 39% (34/87) of the patients in this study achieved pCR. Over all pairs of pathologists, on average 80% of pathology-only predicted pCRs were true pCRs (mean PPV = 80% [range: 69-92%]), and 74% of patients who achieved pCR were predicted pCR by pathology alone (mean sensitivity = 74% [65-82%]). We assessed combinations with MR probability thresholds at PPV levels 50%-70%; and observed the best balance of PPV and sensitivity for the pre-RCB when MR thresholds were set at 50% PPV level. At this threshold setting, the pre-RCB achieved a PPV = 92% [83-100%], meaning on average 92% of predicted pCRs were true pCRs, and this improvement in positive predictive performance over pathology alone is achieved with a lower but still-reasonable 53% sensitivity [33-62%]. Conclusion: Pre-RCB, which predicts a patient as pCR if both MR and inter-regimen pathology predicts pCR, provides clinically actionable accuracy for treatment de-escalation for early responders (PPV>90%). Adding a final MR review at the time of early surgery may further improve performance. Resulting from data presented in this abstract, the pre-RCB algorithm, including the final MR review, has been operationalized and will be used prospectively to identify patients who are highly likely to have already achieved pCR by the inter-regimen timepoint. Citation Format: Sara J Venters, Wen Li, Denise M Wolf, Jodi M Carter, Molly E Klein, Kamaljeet Singh, Kimmie Rabe, I Tolgay Ocal, David Newitt, Christina Yau, Natsuko Onishi, Jessica Gibbs, Sunati Sahoo, Shuko Harada, Laila Khazai, Malini Harigopal, Alexander D Borowsky, Gregor Krings, Ronald Balassanian, Yunn-Yi Chen, Kimberley Cole, Sonal Shad, Barbara LeStage, Amy Delson, Sandra Finestone, Lamorna Brown-Swigart, I-SPY 2 Imaging Working Group, I-SPY 2 TRIAL Consortium, Laura Esserman, Laura van ‘t Veer, W Fraser Symmans, Nola M Hylton. Serial MRI and pathology combined to select candidates for therapy de-escalation in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-10.

Background: The I-SPY 2 TRIAL enrolls women with locally advanced, molecular high-risk breast cancer. An integrated Residual Cancer Burden (iRCB), based on MRI volume change through treatment, is used to predict pathologic complete response (pCR) in the randomization/evaluation Bayesian engine. With the goal of effective de-escalation of treatment for patients exhibiting an early response, biomarkers are being assessed for their ability to predict pCR, alone or with MR data, during treatment. Here, we present the results of a pilot study to examine if invasive tumor cellularity in mid-treatment tissue core biopsies predicts pCR in a 40-patient cohort of I-SPY 2 patients. Other pathologic variables evaluated include Ki67, tumoral histologic features, and stromal tumor-infiltrating lymphocytes (sTILs). Methods: I-SPY 2 TRIAL pathologists (N=4) were provided images of H&E-stained and Ki67 IHC- labelled (DAKO/Agilent, clone MIB-1) core biopsy sections from 40 patients at the inter-regimen time point, ~12-weeks into treatment. Of the 40 patients, 35 had 4 cores, 3 had 3 cores, and 2 had 2 cores assessed. In total, images from 153 cores were evaluated. For each core, pathologists were asked to score the % area occupied by tumor bed (treatment changes and/or residual cancer), % of viable invasive tumor (0-100%) within tumor bed (with Nottingham grading, % Ki67 labelled, and % sTILs, using standardized guidelines). As decided by the pathologist group, only cores with identified tumor bed were included in the initial analysis. Concordance between pathologists was assessed for all scored criteria, using % agreement for dichotomous variables, and Pearson correlation (r)/standard deviation (sd) for continuous variables. The maximum and average cellularity recorded over all cores/patient, averaged over all pathologists, were analyzed for association with pCR using t- tests (significance threshold: p Results: Pathologist were in general agreement about the presence or absence of tumor bed, with greater than 82% agreement between any two (83-96%), and an overall agreement of 77%. For scoring the % of the tumor bed involved by invasive cancer, correlations between pairs of pathologists ranged from 0.79-0.95 (mean(r)=0.87, sd=5%), and agreement on a binary presence/absence of invasive cancer was 78%. Both the mean (t-test: p=7.59E-05) and maximum (t-test: p=0.0012) %invasive tumor at 12 weeks, scored as an average over all pathologists, were significantly higher in patients who did not achieve pCR than in responders. We also treated %invasive cellularity as a dichotomous variable (present/absent). 90% (9/10) of patients scored by all pathologists as 0% invasive tumor cells (absent) achieved a pCR, vs only 20% (6/30) of patients scored as >0% invasive cellularity by one or more pathologists (present) (OR=32, Fisher p=0.0005); yielding a positive predictive value for pCR of 0.9. Ki67 and sTILS at 12 weeks were fairly concordant across pathologists ((r,sd)=(0.92, 8.45%) and (0.82,5.5%), respectively), but did not associate with response (p>0.05 for pCR, RCB01, or RCB index). Tumor histologic grade at 12 weeks, assessed in 29/30 patients with non-zero cellularity, trended toward association (Fisher p=0.078): 44% (4/9) with Grade 3 went on to have a pCR, vs. 15% (2/13) with Grade 2 and 0 with Grade 1. These data demonstrate the utility of invasive tumor cellularity as a predictor of pCR in a clinical setting. Conclusion: In this pilot study we demonstrate that the absence of invasive cancer cells within identified tumor bed in mid-treatment core biopsy samples is highly predictive of pCR. Citation Format: Sara J Venters, Denise M Wolf, Lamorna Brown-Swigart, Christina Yau, Amy L Delson, Bev Parker, Ron Balassanian, Jodi Carter, Yunn-Yi Chen, Kimberly Cole, Laila Khazai, Molly Klein, Dina Kokh, Gregor Krings, Sunati Sahoo, Jane Wei, I-SPY 2 TRIAL Consortium, Laura J Esserman, Laura J van't Veer, W Fraser Symmans. Assessing biomarkers to inform treatment de-escalation: Mid-treatment biopsy cellularity predicts pCR 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 P6-10-02.

Background: Previous studies have independently validated the prognostic relevance of residual cancer burden (RCB) after neoadjuvant chemotherapy. We used results from several independent cohorts in a pooled patient-level analysis to evaluate the relationship of RCB with long-term prognosis across different phenotypic subtypes of breast cancer, to assess generalisability in a broad range of practice settings. Methods: In this pooled analysis, 12 institutes and trials in Europe and the USA were identified by personal communications with site investigators. We obtained participant-level RCB results, and data on clinical and pathological stage, tumour subtype and grade, and treatment and follow-up in November, 2019, from patients (aged ≥18 years) with primary stage I–III breast cancer treated with neoadjuvant chemotherapy followed by surgery. We assessed the association between the continuous RCB score and the primary study outcome, event-free survival, using mixed-effects Cox models with the incorporation of random RCB and cohort effects to account for between-study heterogeneity, and stratification to account for differences in baseline hazard across cancer subtypes defined by hormone receptor status and HER2 status. The association was further evaluated within each breast cancer subtype in multivariable analyses incorporating random RCB and cohort effects and adjustments for age and pretreatment clinical T category, nodal status, and tumour grade. Kaplan-Meier estimates of event-free survival at 3, 5, and 10 years were computed for each RCB class within each subtype. Findings: We analysed participant-level data from 5161 patients treated with neoadjuvant chemotherapy between Sept 12, 1994, and Feb 11, 2019. Median age was 49 years (IQR 20–80). 1164 event-free survival events occurred during follow-up (median follow-up 56 months [IQR 0–186]). RCB score was prognostic within each breast cancer subtype, with higher RCB score significantly associated with worse event-free survival. The univariable hazard ratio (HR) associated with one unit increase in RCB ranged from 1·55 (95% CI 1·41–1·71) for hormone receptor-positive, HER2-negative patients to 2·16 (1·79–2·61) for the hormone receptor-negative, HER2-positive group (with or without HER2-targeted therapy; p

Importance Residual cancer burden (RCB) distributions may improve the interpretation of efficacy in neoadjuvant breast cancer trials. Objective To compare RCB distributions between randomized control and investigational treatments within subtypes of breast cancer and explore the relationship with survival. Design, Setting, and Participants The I-SPY2 is a multicenter, platform adaptive, randomized clinical trial in the US that compares, by subtype, investigational agents in combination with chemotherapy vs chemotherapy alone in adult women with stage 2/3 breast cancer at high risk of early recurrence. Investigational treatments graduated in a prespecified subtype if there was 85% or greater predicted probability of higher rate of pathologic complete response (pCR) in a confirmatory, 300-patient, 1:1 randomized, neoadjuvant trial in that subtype. Evaluation of a secondary end point was reported from the 10 investigational agents tested in the I-SPY2 trial from March 200 through 2016, and analyzed as of September 9, 2020. The analysis plan included modeling of RCB within subtypes defined by hormone receptor (HR) andERBB2 status and compared control treatments with investigational treatments that graduated and those that did not graduate. Interventions Neoadjuvant paclitaxel plus/minus 1 of several investigational agents for 12 weeks, then 12 weeks of cyclophosphamide/doxorubicin chemotherapy followed by surgery. Main Outcomes and Measures Residual cancer burden (pathological measure of residual disease) and event-free survival (EFS). Results A total of 938 women (mean [SD] age, 49 [11] years; 66 [7%] Asian, 103 [11%] Black, and 750 [80%] White individuals) from the first 10 investigational agents were included, with a median follow-up of 52 months (IQR, 29 months). Event-free survival worsened significantly per unit of RCB in every subtype of breast cancer (HR-positive/ERBB2-negative: hazard ratio [HZR], 1.75; 95% CI, 1.45-2.16; HR-positive/ERBB2-positive: HZR, 1.55; 95% CI, 1.18-2.05; HR-negative/ERBB2-positive: HZR, 2.39; 95% CI, 1.64-3.49; HR-negative/ERBB2-negative: HZR, 1.99; 95% CI, 1.71-2.31). Prognostic information from RCB was similar from treatments that graduated (HZR, 2.00; 95% CI, 1.57-2.55; 254 [27%]), did not graduate (HZR, 1.87; 95% CI, 1.61-2.17; 486 [52%]), or were control (HZR, 1.79; 95% CI, 1.42-2.26; 198 [21%]). Investigational treatments significantly lowered RCB in HR-negative/ERBB2-negative (graduated and nongraduated treatments) andERBB2-positive subtypes (graduated treatments), with improved EFS (HZR, 0.61; 95% CI, 0.41-0.93) in the exploratory analysis. Conclusions and Relevance In this randomized clinical trial, the prognostic significance of RCB was consistent regardless of subtype and treatment. Effective neoadjuvant treatments shifted the distribution of RCB in addition to increasing pCR rate and appeared to improve EFS. Using a standardized quantitative method to measure response advances the interpretation of efficacy. Trial Registration ClinicalTrials.gov Identifier:NCT01042379

ObjectivesIn this paper, we discuss leveraging cloud-based platforms to collect, visualize, analyze, and share data in the context of a clinical trial. Our cloud-based infrastructure, Patient Repository of Biomolecular Entities (PRoBE), has given us the opportunity for uniform data structure, more efficient analysis of valuable data, and increased collaboration between researchers.Materials and MethodsWe utilize a multi-cloud platform to manage and analyze data generated from the clinical Investigation of Serial Studies to Predict Your Therapeutic Response with Imaging And moLecular Analysis 2 (I-SPY 2 TRIAL). A collaboration with the Institute for Systems Biology Cancer Gateway in the Cloud has additionally given us access to public genomic databases. Applications to I-SPY 2 data have been built using R Shiny, while leveraging Google's BigQuery tables and SQL commands for data mining.ResultsWe highlight the implementation of PRoBE in several unique case studies including prediction of biomarkers associated with clinical response, access to the Pan-Cancer Atlas, and integrating pathology images within the cloud. Our data integration pipelines, documentation, and all codebase will be placed in a Github repository.Discussion and conclusionWe are hoping to develop risk stratification diagnostics by integrating additional molecular, magnetic resonance imaging, and pathology markers into PRoBE to better predict drug response. A robust cloud infrastructure and tool set can help integrate these large datasets to make valuable predictions of response to multiple agents. For that reason, we are continuously improving PRoBE to advance the way data is stored, accessed, and analyzed in the I-SPY 2 clinical trial.

222 Background: As new tools for prognostic prediction and assessment of treatment response in breast cancer are developed, there is growing interest in treatment optimization strategies to reduce toxic therapy or add additional therapy as needed. The I-SPY 2 trial utilizes a new predictive tool “PreRCB,” which includes mid-treatment MRI and core biopsy responses, to identify patients likely to have a pCR (pathologic complete response) who can de-implement, or forgo, standard-of-care anthracycline-based chemotherapy (AC) after treatment with a taxane + investigational agent. This study assesses initial de-implementation eligibility and selection within the I-SPY2 trial. Methods: All patients enrolled in I-SPY2 since September of 2020 (when the preRCB protocol was first introduced to the trial) who completed the first treatment regimen were evaluated. Patients were only considered for de-implementation of AC if the mid-treatment biopsy was routed to site clinical pathology for review for residual invasive disease. In these patients, preRCB was determined to be predictive of pCR if there was 1) no invasive cancer on the core biopsy and 2) MRI findings met subtype-specific functional tumor volume criteria. Patients meeting criteria for predicted pCR could be offered the option to go directly to surgery by their provider, foregoing pre-operative AC, with the option to receive AC post-operatively should the surgical specimen reveal residual disease. The following outcomes (n,%) were descriptively evaluated: patients participating in I-SPY2, sites with at least one patient evaluated for de-implementation using pre-RCB, patients eligible for de-implementation, patients with surgical RCB assessment, and patients who de-implemented chemotherapy. Results: From September 2020 – May 2021, 145 patients were eligible for analysis. 9/20 (45%) sites with eligible I-SPY patients had at least one patient considered for preRCB assessment. Only 46 patients (32%) had clinical reads on their biopsies, as required for preRCB evaluation. Of these, 48% (14/29) of patients met both MRI and pathology criteria to de-implement AC. However, only seven (50%) of these patients chose to proceed to surgery without preoperative AC. Of those who did not have a predicted pCR using preRBC criteria, 87.5% (28/32) received AC as recommended by the protocol and 12.5% (4/32) did not receive preoperative AC. Conclusions: These pilot results demonstrate slow uptake of this strategy by patients and clinicians. We are currently interviewing patients and physicians to identify and assess facilitators and barriers for patients and physicians to pursue response-guided treatment regimes. Understanding these motivations will create the opportunity to engage site, providers, and patients to engage in research optimizing chemotherapy treatment. Clinical trial information: NCT01042379.

PurposeIn the on-going I-SPY2 TRIAL, participants receive 12 cycles of weekly paclitaxel with or without addition of experimental agents for 12 weeks (first regimen), followed by 4 cycles of anthracycline-cyclophosphamide (AC, second regimen) prior to surgery. A de-escalation strategy currently being introduced in I-SPY2 will give patients the option to skip AC if it is highly likely that they have achieved early pathologic complete response (pCR) at the inter-regimen time point (12 weeks). To guide selection of candidates for this option, a prediction model combining subtype-specific MRI predictive probabilities with mid-treatment percutaneous core biopsy pathology has been developed. The combined model predicts a patient as early pCR if the MRI model based on a quantitative measure of functional tumor volume predicts a high likelihood of pCR, and pathology determines that there are no invasive cancer cells detected in the mid-treatment core biopsy. This study was performed to develop radiologic criteria for post-selection review to further reduce the possibility for incorrect de-escalation recommendation. MethodsA review of 87 I-SPY2 patients with serial MRI and inter-regimen core biopsy identified 25 patients where both the subtype specific MRI model and at least 2 of 11 I-SPY2 pathologists predicted early pCR. One radiologist retrospectively reviewed MRIs at pre-treatment and inter-regimen for the 25 patients in a blinded fashion, and labeled the presence of residual disease on MRI at inter-regimen as follows: rank 0, no residual disease; rank 1, possible residual disease; rank 2, obvious residual disease.To evaluate the accuracy of selecting candidates for de-escalation of neoadjuvant therapy (i.e. predicting patients with early pCR at inter-regimen), surgical pathology of pCR after the completion of NAC (both first and second regimens) was defined as a surrogate “truth” in this study. Positive predictive value (PPV) and sensitivity for the combined MRI-pathology model were computed for all pathologist pairs. After adding qualitative radiologic review, patients labeled as rank 2 were excluded from the candidates with predicted early pCR by the combined MRI-pathology model. PPV and sensitivity before and after radiologic review were compared. ResultsThe 25 patients included 21 patients that ultimately achieved pCR, and 4 patients that did not. Radiologic review of the inter-regimen MRI for these patients classified their MRIs as 20% (5/25) rank 0 (no residual disease), 36% (9/25) rank 1 (possible residual disease), and 44% (11/25) rank 2 (obvious residual disease). Most (3/4) non-pCR patients were classified rank 2, however one (1/4) was assessed rank 0. Eight of the 21 pCR patients (38%) were classified as having obvious residual disease on inter-regimen MRI. Without radiologic review, the combined MRI-pathology model predicted pCR with a mean PPV of 92% (range 83-100%) and a mean sensitivity of 91% (range 76-100%). When patients labeled as rank 2 by radiologic review were excluded from candidates with predicted early pCR, PPV increased to 99% (range 93-100%) at the expense of lower sensitivity of 59% (range 52-62%). Conclusion In this study, elimination of rank 2 lesions resulted in substantial improvement in PPV (92% to 99%) with a consequent reduction in sensitivity from 91% to 59%. Notably, none of the rank 1 lesions, where diagnostic interpretation was less certain, were found to be non-pCR at surgery. High PPV is essential to ensure high accuracy and safety in directing early pCR patients to therapy de-escalation. These findings will be further validated in continuing studies and radiologic re-review will be included in the de-escalation strategy for omission of AC in the I-SPY2 TRIAL. Citation Format: Natsuko Onishi, Wen Li, Sara J Venters, Denise M Wolf, David C Newitt, Elissa R Price, Jessica Gibbs, Barbara LeStage, the I-SPY 2 Pathology Working Group, the I-SPY 2 Imaging Working Group, the I-SPY 2 Consortium, William F Symmans, Nola M Hylton. Radiologic review to refine selection of candidates for de-escalation of neoadjuvant therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-02.

Background: During development of the vertebrate eye, optic tissue is progressively compartmentalized into functionally distinct tissues. From the central to the peripheral optic cup, the original optic neuroepithelial tissue compartmentalizes, forming retina, ciliary body, and iris. The retina can be further sub-divided into peripheral and central compartments, where the central domain is specialized for higher visual acuity, having a higher ratio and density of cone photoreceptors in most species. Results: Classically, models depict a segregation of the early optic cup into only two domains, neural and non-neural. Recent studies, however, uncovered discrete precursors for central and peripheral retina in the optic vesicle, indicating that the neural retina cannot be considered as a single unit with homogeneous specification and development. Instead, central and peripheral retina may be subject to distinct developmental pathways that underlie their specialization. Conclusions: This review focuses on lineage relationships in the retina and revisits the historical context for segregation of central and peripheral retina precursors before overt eye morphogenesis. Developmental Dynamics 244:266–276, 2015. © 2014 Wiley Periodicals, Inc.

ctDNA analysis offers a non-invasive approach for monitoring response and resistance to treatment. Serial ctDNA testing during neoadjuvant therapy (NAT) may provide early indicators of emerging resistance and disease progression. In this study, we analyzed ctDNA from high-risk early breast cancer patients who received NAT and definitive surgery in the I-SPY 2 TRIAL (NCT01042379). We hypothesize that (1) assessment of ctDNA levels early in treatment will improve the performance of molecular and imaging-based predictors of pathologic complete response (pCR) to NAT; and (2) levels of ctDNA after NAT are associated with residual cancer burden and recurrence [distant recurrence free survival (DRFS)]. Methods: ctDNA analysis was performed in 84 high-risk stage II and III breast cancer patients randomized to neoadjuvant investigational agent (n=52), AKT inhibitor MK-2206 (M) in combination with paclitaxel (T) followed by doxorubicin and cyclophosphamide (AC) (M+T->AC), or standard-of-care (T->AC) (n=32). HER2+ patients also received trastuzumab (H). Serial plasma was collected before NAT (T0), early treatment (3 weeks, T1), between regimens (12 weeks, T2), and after NAT prior to surgery (T3). Mutational profiles derived from pretreatment tumor biopsy and normal matched DNA whole exome sequencing were used to design personalized assays targeting 16 patient-specific somatic variants to detect ctDNA in serial plasma. Results: Of the 84 patients in this study, 43% were HR-/HER2- (TNBC), 35% HR+/HER2-, and 23% HER2+. In total, 74% (61 of 83), 35% (28 of 79), 14% (9 of 65), and 8% (5 of 61) were positive for ctDNA at timepoints T0, T1, T2, and T3, respectively. At T0, ctDNA positivity and levels (average number of mutant molecules detected per mL) were significantly associated with increased tumor burden (by clinical and MRI examination), more aggressive tumor biology (as reflected in higher Mammaprint scores and grade) and subtype (HER2+ and TNBC). Twenty-seven percent (27%) of the 84 patients achieved a pCR and all patients who were ctDNA-positive at T3 (n=5) did not achieve a pCR. Currently, data are being collected to: (1) assess the relationship of ctDNA and MRI imaging in predicting tumor response to therapy; (2) examine the relationship of ctDNA levels before and after NAT with 3-year DRFS and event-free survival (EFS). The results of these analyses will be presented at the SABCS 2018 meeting. Conclusions: Our study provides a platform to evaluate the clinical significance of ctDNA for serial monitoring of response to NAT. Accurate and early response prediction by highly sensitive ctDNA analysis can facilitate a timely and judicious change in treatment to improve patients' chances of achieving a pCR. Finally, personalized ctDNA testing may complement imaging and pathologic evaluation of tumor response to fine-tune pCR as a surrogate endpoint for improved DRFS and EFS. Citation Format: Magbanua MJM, Brown-Swigart L, Hirst GL, Yau C, Wolf D, Ma AA, Bergin E, Venters S, Sethi H, Wu H-T, Salari R, Tin T, Sawyer S, Louie M, Zimmermann B, Lin C-HJ, Keats J, Liang WS, Cuyugan L, Enriquez D, Tripathy D, Chien AJ, Forero A, DeMichele A, Liu M, Delson AL, Asare S, Esserman L, van't Veer L, I-SPY 2 Consortium. Personalized serial circulating tumor DNA (ctDNA) analysis in high-risk early stage breast cancer patients to monitor and predict response to neoadjuvant therapy and outcome in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-01.

The timing of myogenic differentiation of hypaxial muscle precursor cells in the somite lags behind that of epaxial precursors. Two hypotheses have been proposed to explain this delay. One attributes the delay to the presence of negative-acting signals from the lateral plate mesoderm adjacent to the hypaxial muscle precursor cells located in the ventrolateral lip of the somitic dermomyotome (Pourquié et al. [1995] Proc. Natl. Acad. Sci. USA 92:3219-3223). The second attributes the delay to an absence of positive-acting inductive signals, similar to those from the axial structures that induce epaxial myotome development (Pownall et al. [1996] Development 122:1475-1488). Because both studies relied principally upon changes in the expression pattern of mRNAs specific to early muscle precursor cell markers, we revisited these experiments using two methods to assess muscle terminal differentiation. First, injection of fluorescent dyes before surgery was used to determine whether ventrolateral lip cells transform from epithelial cells to elongated myocytes. Second, an antibody to a terminal differentiation marker and a new monoclonal antibody that recognises avian and mammalian Pax3 were used for immunohistochemistry to assess the transition from precursor cell to myocyte. The results support both hypotheses and show further that placing axial structures adjacent to the somite ventrolateral lip induces an axial pattern of myocyte terminal differentiation and elongation.

During development of the vertebrate eye, optic tissue is progressively compartmentalized into functionally distinct tissues. From the central to the peripheral optic cup, the original optic neuroepithelial tissue compartmentalizes, forming retina, ciliary body and iris. The retina can be further sub-divided into peripheral and central compartments, where the central domain is specialized for higher visual acuity, having a higher ratio and density of cone photoreceptors in most species. Classically, models depict a segregation of the early optic cup into only two domains, neural and non-neural. Recent studies, however, uncovered discrete precursors for central and peripheral retina in the optic vesicle, indicating that the neural retina cannot be considered as a single unit with homogeneous specification and development. Instead, central and peripheral retina may be subject to distinct developmental pathways that underlie their specialization. This review focuses on lineage relationships in the retina and revisits the historical context for segregation of central and peripheral retina precursors before overt eye morphogenesis.

The morphogenetic cell movements responsible for growth and morphogenesis in vertebrate embryos are poorly understood. Myotome precursor cells undergo myotomal translocation; a key morphogenetic cell movement whereby myotomal precursor cells leave the dermomyotome epithelium and enter the subjacent myotome layer where myogenic differentiation ensues. The precursors to the embryonic epaxial myotome are concentrated in the dorsomedial lip (DML) of the somite dermomyotome (W. F. Denetclaw, B. Christ and C. P. Ordahl (1997) Development 124, 1601-1610), a finding recently substantiated through surgical transplantation studies (C. P. Ordahl, E. Berdougo, S. J. Venters and W. F. Denetclaw, Jr (2001) Development 128, 1731-1744). Confocal microscopy was used here to analyze the location and pattern of myotome cells whose precursors had earlier been labeled by fluorescent dye injection into the middle region of the DML, a site that maximizes the potential to discriminate among experimental outcomes. Double-dye injection experiments conducted at this site demonstrate that cells fated to form myotome do not involute around the recurved epithelium of the DML but rather are displaced laterally where they transiently intermingle with cells fated to enter the central epithelial sheet region of the dermomyotome. Time- and position-dependent labeling experiments demonstrated that myotome precursor cells translocate directly from the middle region of the DML without prior intra-epithelial ‘translational’ movements of precursor cells to either the cranial or caudal lips of the dermomyotome epithelium, nor were any such translational movements evident in these experiments. The morphogenetic cell movements demonstrated here to be involved in the directional growth and segmental patterning of the myotome and dermomyotome bear interesting similarities with those of other morphogenetic systems.

The structure and development of the myotome has been extensively studied in birds and amphibians but few studies have systematically addressed its development in mammals. We have used a transgenic mouse carrying an nLacZ marker coupled to a myosin light chain 3F promoter to describe the structure of the developing mammalian myotome. Through studies of transgene expression pattern, coupled with immunohistochemistry for the muscle structural proteins desmin and slow myosin heavy chain we describe a gradient of maturity for the cells within the developing myotome. Our results show that the earliest myocytes of the mammalian myotome span the rostrocaudal extent of the somite and have single large nuclei which localise centrally within the myotome. Throughout the period of study the myotome is more mature ventrally than dorsally and cells comprising the medial aspect of the myotome are younger than those lying laterally. Immunohistochemistry for the earliest expressed muscle regulatory factor (myf-5) is used to define areas of the myotome contributing new myogenic cells. In the early myotome small, round, myf-5-expressing cells are found extensively within the dorsomedial aspect of the dermamyotome and also within the entire rostral and caudal dermamyotomal lips. They subsequently appear within the central zone of the myotome, adjacent to the medially curled rostral and caudal dermamyotomal lips, and there begin to elongate symmetrically. As the myotome enlarges, myf-5 expression is always restricted to the most medial aspect of the myotome, adjacent to the least mature myocytes, marking the site of addition of new myogenic cells. Together, these results allow development of a model of mammalian myotome formation where growth occurs medially by addition of new cells from both rostral and caudal dermamyotome lips, while more mature myocytes are displaced laterally. Furthermore, early myotomal myocytes differentiate in the absence of MyoD expression, unlike later myotomal myocytes. This, along with their distinct morphology, suggests these cells may form a separate lineage of pioneer myogenic cells. Dev Dyn 1999;216:219–232. ©1999 Wiley-Liss, Inc.

In the mature eye, three distinct tissue fates, retina, ciliary body, and iris, arrange with a strict linear organization along the central (back) to peripheral (front) axis. The establishment of this topographical relationship within the optic vesicle is not well understood. We use a targeted vital labeling strategy to test the derivation of mature eye tissues from the optic vesicle of the chick embryo. Fate mapping uncovers two distinct origins of the neural retina. Contrary to expectations, the central neural retina has a discrete origin within the posterior optic vesicle. The peripheral retina derives from the distal optic vesicle, sharing a common origin with more peripheral tissue fates. This study identifies for the first time two distinct retinal sub-domains, central and peripheral, which arise during embryogenesis. Identification of these discrete retinal compartments provides a framework for understanding functional and disease processes throughout retinal tissue.

Phase specificity, the temporal and tissue restriction of teratogen-induced defects during embryonic -development, is a poorly understood but common property of teratogens, an important source of human birth defects. Somite counting and somite units are novel chronometric tools used here to identify stages of paraxial mesoderm development that are sensitive to pulse-chase exposure (2 to16 h) to 5-bromodeoxyuridine (BrdU). In all cases, it was the presomitic mesoderm (PSM) that was sensitive to BrdU induced segmentation anomalies. At high concentration (1.0 × 10(-2) M BrdU), PSM presegment stages ss-IV and earlier were irreversibly inhibited from completing segmentation. At low concentration (2.6 × 10(-6) M), BrdU induced periodic focal defects that predominantly trace back to PSM presegments between ss-V and ss-IX. Phase specificity is characteristic of both types of segmentation anomalies. Focal segmentation defects are phase-specific because they result from disruption of 2-3 presegments in the PSM while adjacent -rostral and caudal presegments are (apparently) unaffected. Irreversible inhibition of segmentation is also phase-specific because only PSM presegments ss-IV or earlier were affected while older segments (ss-III to ss-I) were able to complete segmentation. The presegments predominantly affected have not yet passed the determination front, the point at which the segmentation clock establishes somite rostro-caudal -polarity. Somite unit chronometry provides a means to identify specific PSM presegment stages that are susceptible to induced segmentation defects and the biological processes that underlie that vulnerability.

Purpose The optic cup is created through invagination of the optic vesicle. The morphogenetic rearrangement creates a double-layered cup, with a hinge (the Optic Cup Lip) where the epithelium bends back upon itself. Shortly after the optic cup forms, it is thought to be sub-divided into separate lineages: i) pigmented epithelium in the outer layer; ii) presumptive iris and ciliary body at the most anterior aspect of the inner layer; and iii) presumptive neural retina in the remainder of the inner layer. We test the native developmental potential of the anterior cup to determine if it normally contributes to the retina. Methods Vital dye and green fluorescent protein (GFP) expressing replication-incompetent retroviral vectors were used to label cells in the nascent optic cup and follow their direct progeny throughout development. Label was applied to either the optic cup lip (n=40), or to the domain just posterior to the lip (n=20). Retroviral labeling is a permanent lineage marker and enabled the analysis of advanced stages of development. Results Labeling within the optic cup gave rise to labeled progeny in the posterior optic cup that differentiated as neural retina (20 of 20). In contrast, labeling cells in the optic cup lip gave rise to progeny of labeled cells arrayed in a linear progression, from the lip into the neural retina (36 of 40). Label was retained in cells at the optic cup lip, regardless of age at examination. In older embryos, labeled progeny delaminated from the optic cup lip to differentiate as muscle of the pupillary margin. Conclusions The data show that the cells at the optic cup lip are a common progenitor population for pigmented epithelium, anterior eye tissues (ciliary body, iris, and pupillary muscle) and retinal neurons. The findings are supportive of an interpretation where the optic cup lip is a specialized niche containing a multipotent progenitor population.

Phase specificity, the temporal and tissue restriction of teratogen-induced defects during embryonic -development, is a poorly understood but common property of teratogens, an important source of human birth defects. Somite counting and somite units are novel chronometric tools used here to identify stages of paraxial mesoderm development that are sensitive to pulse-chase exposure (2 to >16 h) to 5-bromodeoxyuridine (BrdU). In all cases, it was the presomitic mesoderm (PSM) that was sensitive to BrdU induced segmentation anomalies. At high concentration (1.0 × 10(-2) M BrdU), PSM presegment stages ss-IV and earlier were irreversibly inhibited from completing segmentation. At low concentration (2.6 × 10(-6) M), BrdU induced periodic focal defects that predominantly trace back to PSM presegments between ss-V and ss-IX. Phase specificity is characteristic of both types of segmentation anomalies. Focal segmentation defects are phase-specific because they result from disruption of 2-3 presegments in the PSM while adjacent -rostral and caudal presegments are (apparently) unaffected. Irreversible inhibition of segmentation is also phase-specific because only PSM presegments ss-IV or earlier were affected while older segments (ss-III to ss-I) were able to complete segmentation. The presegments predominantly affected have not yet passed the determination front, the point at which the segmentation clock establishes somite rostro-caudal -polarity. Somite unit chronometry provides a means to identify specific PSM presegment stages that are susceptible to induced segmentation defects and the biological processes that underlie that vulnerability.

We describe two replication incompetent retroviral vectors that co-express green fluorescent protein (GFP) and beta-galactosidase. These vectors incorporate either the avian reticuloendotheliosis (spleen necrosis virus; SNV) promoter or the chick beta-actin promoter, into the backbone of the murine leukemia (MLV) viral vector. the additional promoters drive transgene expression in avian tissue. the remainder of the vector is MLV-like, allowing high titer viral particle production by means of transient transfection. the SNV promoter produces high and early expression of introduced genes, enabling detection of the single copy integrated GFP gene in infected cells and their progeny in vivo. Substitution of the LacZ coding DNA with a relevant gene of interest will enable its co-expression with GFP, thus allowing visualization of the effect of specific and stable changes in gene expression throughout development. As the VSV-G pseudotyped viral vector is replication incompetent, changes in gene expression can be controlled temporally, by altering the timing of introduction. Developmental Dynamics 237.3260-3269, 2008. Published 2008 Wiley-Liss, Inc. Univ Calif San Francisco, Dept Neurosurg, San Francisco, CA 94143 USA Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, Brazil Web of Science

Somite stages were employed as units of intrinsic developmental time to measure cell doubling rate and other cell cycle parameters of chick forelimb level somites. Somite cell nuclei doubled over an interval corresponding to approximately 7+ somite stages (7+ ss; approximately 11 hr) and approximately 24 new primary myotome cells are born per somite stage ( approximately 16/hr). FACS analysis of DNA content in dissociated paraxial mesoderm cells indicated that slightly more than half are in G1/G0 phase of the cell cycle and that the average combined length of the S phase and G2 phase intervals is approximately 3 ss ( approximately 4.5 hr). A wavefront of increased mitotic nuclei per segment coincident with somite budding potentially reflects a surge in the number of cells entering S phase 3 ss earlier as each PSM segment becomes unresponsive to FGF signaling as it passes through the determination front.

To determine if somitic stem cell pools could be identified by an intrinsic difference in mitotic behaviour, the orientation of mitoses in the dermomyotome epithelium was analysed. We describe a concentration of apico-basal mitoses within the dermomyotome dorsomedial lip (DML). The occurrence of apico-basal divisions is closely associated with asymmetric localisation of the notch pathway factor numb, allowing description of such divisions as asymmetric. In contrast, planar divisions, occurring in the plane of the epithelium, are symmetric. Further, we show that the DML environmental niche is sufficient to promote numb expression in epaxial dermomyotome tissue that does not normally express this factor. These data provide, for the first time, a non-retrospective tracing analysis of the mechanism by which the DML fulfils the stem-cell pool role it plays during epaxial primary myotome morphogenesis.

The cellular and molecular mechanisms that govern early muscle patterning in vertebrate development are unknown. The earliest skeletal muscle to organize, the primary myotome of the epaxial domain, is a thin sheet of muscle tissue that expands in each somite segment in a lateral-to-medial direction in concert with the overlying dermomyotome epithelium. Several mutually contradictory models have been proposed to explain how myotome precursor cells, which are known to reside within the dermomyotome, translocate to the subjacent myotome layer to form this first segmented muscle tissue of the body. Using experimental embryology to discriminate among these models, we show here that ablation of the dorsomedial lip (DML) of the dermomyotome epithelium blocks further primary myotome growth while ablation of other dermomyotome regions does not. Myotome growth and morphogenesis can be restored in a DML-ablated somite of a host embryo by transplantation of a second DML from a donor embryo. Chick-quail marking experiments show that new myotome cells in such recombinant somites are derived from the donor DML and that cells from other regions of the somite are neither present nor required. In addition to the myotome, the transplanted DML also gives rise to the dermomyotome epithelium overlying the new myotome growth region and from which the mesenchymal dermatome will later emerge. These results demonstrate that the DML is a cellular growth engine that is both necessary and sufficient to drive the growth and morphogenesis of the primary myotome and simultaneously drive that of the dermomyotome, an epithelium containing muscle, dermis and possibly other potentialities.