Residual Disease In Breast Cancer Research Articles

Flower-Like Nanosensors for Photoacoustic-Enhanced Lysosomal Escape and Cytoplasmic Marker-Activated Fluorescence: Enabling High-Contrast Identification and Photothermal Ablation of Minimal Residual Disease in Breast Cancer.

The clearance of minimal residual disease (MRD) after breast cancer surgery is crucial for inhibiting metastasis and recurrence. However, the most promising biomarker-activated fluorescence imaging strategies encounter accessibility issues of the delivered sensors to cytoplasmic targets. Herein, a flower-like composite nanosensor with photoacoustic (PA) effect-enhanced lysosomal escape and cytoplasmic marker-activated fluorescence is developed to address this challenge. Specifically, the incorporation of Co2+ into the synthesis of 2D Zn2+-derived metal-organic frameworks enabled rapid dopamine polymerization and deposition. Subsequently, the composite nanoflower (FHN), characterized by an average size of ≈80 nm and petal thickness of ≈6 nm, is formed through the sealing of micropores and simultaneous cross-linking of nanosheets. The pronounced reduction in thermal conductivity of FHN, and superposition of interpetal thermal fields under a pulsed laser (PL), lead to enhanced PA effect and membrane permeability. Thereby, nanosensors efficiently escape from lysosomes resulting in synergistic fluorescence activation by dual-factors (ATP, miRNA-21) and DNA probes installed on FHN. A subsequently high tumor-to-normal tissue signal ratio (TNR) of 17.4 lead to precise guidance of NIR irradiation for efficient MRD eradication and recurrence inhibition. This study provides a new approach for high-contrast identification and precise ablation of MRD based on the synergistic response of endogenous and exogenous factors.

Knowledge-map and research trends of circulating tumor cells in breast cancer: a scientometric analysis

Assessing circulating tumor cells (CTCs) in early-stage breast cancer patients can help identify relapse risk for timely interventions. Molecular analysis of CTCs can reveal vulnerabilities for personalized treatment options in metastatic breast cancer. This study aims to summarize CTCs in breast cancer research understanding and evaluate research trends. Extracted from the Web of Science Core Collection, publications on CTCs in breast cancer studies spanning from January 1, 2008, to December 21, 2023, were included. Co-authorships, references, and keywords were analyzed using Bibliometrix R packages and VOSviewer software. References and keywords burst detection were conducted with CiteSpace, and BICOMB was utilized to generate high-frequency keyword layouts. Biclustering analysis of the binary co-keyword matrix was performed using gCLUTO. 1747 articles focusing on CTCs in breast cancer were identified. The USA and the University of Texas MD Anderson Cancer Center demonstrated the highest productivity at the national and institutional levels, respectively. The journal “CANCERS” had the highest publication outputs on this subject. Pantel K emerged as the foremost author with the highest publication and co-citation counts. Analysis of co-keywords unveiled five prominent research areas concerning CTCs in breast cancer. The prognostic and predictive roles of CTCs in breast cancer have substantial implications for clinical practice. Nevertheless, precise assessment of CTCs, encompassing its quantities and attributes through advanced technologies, and its role in detecting minimal residual disease in breast cancer, continue to pose notable challenges. In conclusion, recent advancements and trends in CTCs research in breast cancer are examined through scientometric analysis in this study. The results provide valuable insights for the formulation of novel approaches in CTCs research, emphasizing the current research frontiers.

Abstract PO2-14-04: Circulating tumor DNA after neoadjuvant chemotherapy is a better prognostic test than Residual Cancer Burden in patients with triple negative breast cancer and residual tumor

Abstract Background The presence of residual tumor at surgery (non-pathological complete response or non-pCR) occurs in about half of TNBCs treated with neoadjuvant chemotherapy (NAC) signals chemoresistance. Although the addition of further adjuvant chemotherapy (Capecitabine/Xeloda) significantly improves RFS in these patients, the majority of patients nevertheless have a good prognosis, and thus may not require adjuvant Capecitabine. One of the best prognostic biomarkers in these patients is Residual Cancer Burden (RCB), which relies on the pathological examination of the residual tumor bed and lymph nodes obtained at surgery. Circulating tumor DNA (ctDNA) is a plasma-based biomarker, which has been shown to accurately detect minimal residual disease in breast cancer. We have previously shown that detection of ctDNA after NAC but before surgery in patients with non-pCR signals very poor prognosis, while non-detectable ctDNA is associated with an excellent prognosis. We now compare ctDNA to RCB score in TNBC patients with non-pCR. Methods In the multicenter TRICIA study, 44 TNBC patients with non-pCR after NAC in 5 hospital centers had consented to serial plasma collection prior to and after surgery. RCB score was calculated from pathology records. Whole exome sequencing (WES) was performed on DNA extracted from primary tumor biopsies or residual tumors at surgery to identify tumor-specific mutations (5 variants/patient). Digital droplet PCR (ddPCR) assays were developed for these mutations and tests were run in an academic hospital setting. Patients with at least one detectable mutation were considered ctDNA positive for a given time point. The detection of ctDNA was correlated with relapse-free survival (RFS). Results: The median follow up was 40 months. 19 of the 44 patients relapsed within 2 years after surgery. Detection of ctDNA at the end of NAC but before surgery (T1) was correlated with very poor RFS (H.R. = 0.15, p< 0.0001 (95% CI = 0.067-0.35)). 14/15 patients (93%) without detectable ctDNA did not recur, while 76% of patients with detectable ctDNA relapsed. The RCB score distribution was as follows: 7 RCB1, 21 RCB2 and 16 RCB3. According to RCB score, 1/7 RCB1, 11/21 RCB2 and 11/16 RCB3 patients relapsed. All but one of the relapsed patients had detectable ctDNA in at least one timepoint. All 11 RCB3 patients that relapsed had detectable ctDNA while none of the 5 RCB3 patients without relapse had detectable ctDNA. The one RCB1 patient with detectable ctDNA also relapsed. In cox regression analysis, the lack of ctDNA detection was significantly associated with excellent RFS in RCB2 (HR=0.16, p=0.003, (95% CI=0.05-.53)) and RCB3 groups (HR=0.10, p=0.0005, 95% CI= 0.03-.36). Conclusion ctDNA testing using ddPCR in an academic hospital-based setting at the post-NAC time point identifies an excellent prognostic group in TNBC patients with non-pCR and can discriminate very good from very poor prognosis in both RCB2 and RCB3 score subgroups. The detection of ctDNA may be superior to RCB scores in assessing prognosis in patients with non-pCR. Citation Format: Mark Basik, Talia Roseshter, Anna Klemantovich, Luca Cavallone, Adriana Aguilar-Mahecha, Josiane Lafleur, Cathy Lan, Oluwadara Elebute, Sarah Jenna, Jean-Francois Boileau, Manuela Pelmus. Circulating tumor DNA after neoadjuvant chemotherapy is a better prognostic test than Residual Cancer Burden in patients with triple negative breast cancer and residual tumor [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-04.

Clinical Applications for Liquid Biopsy Assessment of Minimal Residual Disease in Breast Cancer

Clinical Applications for Liquid Biopsy Assessment of Minimal Residual Disease in Breast Cancer

Abstract PD5-02: PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer

Abstract Background: While new treatments and improved subtyping schemas are anticipated to improve treatment response in triple-negative breast cancer (TNBC) patients, therapeutic resistance remains a significant challenge. Moreover, there is an urgent need for additional research model systems to study resistance and residual disease in breast cancer, including aggressive subtypes of breast cancer. Organoid culture is a promising technology used for growing breast cancer cells with high efficiency; however, the extent to which treatment resistance can be modeled using this system is unknown. This research used patient-derived organoid cultures in the context of computational analyses of large molecular and clinical datasets to study resistance mechanisms, biomarkers, and alternative treatment strategies to overcome drug resistance in early-stage TNBC. Methods: Organoid cultures were derived from breast tumor samples (taken from lumpectomy, mastectomy, or core biopsy samples), digested to the organoid level using collagenase, and grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium (Dekkers et al. Nat Protoc 2021). An evaluation of all available I-SPY2 biomarker data (Wolf et al. Cancer Cell 2022) was performed focusing on genes, proteins, and pathways associated with resistance. These were then used to study whether resistance biomarkers identified in patient tumors are also present in organoids propagated from breast cancer post-treatment residual disease. To this end, bulk RNA sequencing data of organoids were normalized and merged with the TCGA dataset (Hoadley et al. Cell 2018) to enable analysis in a larger context, and immunofluorescence staining of organoids was performed. A high-throughput 386 anti-cancer drug compound screen and subsequent synergy testing with the most promising compounds were performed to identify and predict alternative treatment strategies. Additional assays to explore kinome activity in this organoid model are in progress. Results: A TNBC organoid biobank was established (n=31), which was enriched for inflammatory breast cancer (IBC; n=15), an aggressive form of breast cancer. Most organoids were derived from residual disease after neoadjuvant therapy. Bulk RNA sequencing analysis performed on 10 TNBC organoids revealed 3 subsets that were characterized predominantly by either normal-like/luminal androgen receptor or basal-like features or expressed distinct gene expression profiles, with IBC cases present in all 3 subsets. Intriguingly, the IBC organoids were characterized by higher expression of a number of immune-related signatures, despite an absence of clear immune cells in culture. A residual disease IBC/TNBC organoid resistant to chemotherapy was used to perform the 386-drug compound screen. The organoid model showed resistance to veliparib-cisplatin, consistent with the expression of gene/protein biomarkers predictive of drug resistance found in I-SPY2 (low PARPi7 levels and high pFOXO1 and pMEK1/2 expression). In addition, the screen identified multiple classes of inhibitors as promising synergistic/additive candidates for overcoming resistance to cisplatin. Conclusion: In this proof-of-principle study, we demonstrated the utility of matching I-SPY2 resistance biomarkers and signatures to residual disease tumor organoid cultures. We show that tumor organoid cultures modeling drug resistance states are a useful complement to existing research models of breast cancer and can be used for compound testing. We have developed a pipeline to propagate residual tumors from patients enrolled in I-SPY2 into organoid cultures to create a broader platform for preclinical drug testing informed by tumor biology with the ultimate goal of enabling faster, more successful translational studies and increased treatment options for resistant disease. Citation Format: Tam Binh V. Bui, Denise M. Wolf, Kaitlin Moore, Isaac S. Harris, Pravin Phadatare, Christina Yau, Lamorna A. Brown Swigart, Laura J. Esserman, Jean-Philippe Coppe, Julia Wulfkuhle, Emanuel F. Petricoin, Michael Campbell, Laura M. Selfors, Deborah A. Dillon, Beth Overmoyer, Filipa Lynce, Laura Van ’t Veer, Jennifer Rosenbluth. PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-02.

Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients.

Precisión diagnóstica de la mamografía con contraste vs RM en la valoración de la respuesta del cáncer de mama tratado con quimioterapia neoadyuvante

IntroductionMagnetic resonance imaging is the most accurate diagnostic method for evaluating residual disease in breast cancer treated with neoadjuvant chemotherapy. Contrast-enhanced mammography can be an effective alternative to MRI in the evaluation of residual tumor. This technique would significantly reduce costs, improve patient flow and hardly has any contraindications. MethodsWe have carried out an observational and prospective study in 43 patients with breast cancer treated with neoadjuvant chemotherapy. The pre-surgical study included both mammography with contrast injection and magnetic resonance imaging. Residual tumor size by imaging was correlated with the postoperative pathology study. ResultsThe CM presents a higher interclass correlation coefficient than the RM (0.9 vs. 0.7). The sensitivity and specificity values of CM (83.9% and 83.3%) are high and comparable to those of MRI (74.2% and 91.6%). Furthermore, the negative predictive value of CM is greater than that of MRI (66.7% vs 57.9%) and PPV is very similar (92.9% vs 95.8%). ConclusionContrast-enhanced mammography is a test comparable to magnetic resonance imaging for the evaluation of post-neoadjuvant tumor response in patients with breast cancer. It is also a valid test in visualizing additional lesions in the same or contralateral breast.

Metabolic memory underlying minimal residual disease in breast cancer

Tumor relapse from treatment‐resistant cells (minimal residual disease, MRD) underlies most breast cancer‐related deaths. Yet, the molecular characteristics defining their malignancy have largely remained elusive. Here, we integrated multi‐omics data from a tractable organoid system with a metabolic modeling approach to uncover the metabolic and regulatory idiosyncrasies of the MRD. We find that the resistant cells, despite their non‐proliferative phenotype and the absence of oncogenic signaling, feature increased glycolysis and activity of certain urea cycle enzyme reminiscent of the tumor. This metabolic distinctiveness was also evident in a mouse model and in transcriptomic data from patients following neo‐adjuvant therapy. We further identified a marked similarity in DNA methylation profiles between tumor and residual cells. Taken together, our data reveal a metabolic and epigenetic memory of the treatment‐resistant cells. We further demonstrate that the memorized elevated glycolysis in MRD is crucial for their survival and can be targeted using a small‐molecule inhibitor without impacting normal cells. The metabolic aberrances of MRD thus offer new therapeutic opportunities for post‐treatment care to prevent breast tumor recurrence.

Predicting response to neo-adjuvant chemotherapy and assessment of residual disease in breast cancer using contrast-enhanced spectral mammography: a combined qualitative and quantitative approach

BackgroundMagnetic resonance imaging (MRI) is the gold standard imaging modality for evaluation of response for neo-adjuvant chemotherapy (NAC) in breast cancer as it has the advantage of providing both; morphology assessment together with providing functional information which can be obtained by contrast injection. Until the recent emergence of contrast-enhanced mammography as a promising breast imaging modality, these features were considered unique for MRI. The aim of the study is to evaluate the competence of contrast-enhanced spectral mammography (CESM) in the prediction of response to NAC and the assessment of residual disease extent, as well as the assessment of a new combined (quantitative and qualitative) evaluation approach that is proposed by the authors. The study included 81 patients with pathologically proved breast cancer scheduled for receiving NAC. They underwent 2 CESM examinations; pre- and post-NAC (maximum 10 days before surgery). All patients were assessed using the RECIST 1.1 criteria and a combined approach (RECIST+ qualitative subjective assessment). Results were in correlation to postoperative pathology using the Miller-Payne grading. For statistical evaluation, patients were classified into responders and non-responders.ResultsPostoperative histopathology showed that 60/81 lesions were responders (Miller-Payne grades 3, 4, and 5) while the combined response evaluation approach and RECIST 1.1 alone showed 57/60 (95%) patients and 46/60 patients (76.7%) as responders respectively. The combined response evaluation approach showed higher sensitivity and positive and negative predictive values compared to the evaluation based on RECIST alone (95%, 87.6%, and 81.2% compared to 76.6%, 86.7%, and 50% respectively).ConclusionCESM can be readily used to assess tumor response to NAC and allows the assessment of functional changes in residual tumor cells in addition to size discrepancy. Using CESM, we could accurately assess residual tumor extent and thus enable better surgical planning.

Quantitative mathematical objective evaluation of contrast-enhanced spectral mammogram in the assessment of response to neoadjuvant chemotherapy and prediction of residual disease in breast cancer

BackgroundThe aim of the study is to initiate a new quantitative mathematical objective tool for evaluation of response to neoadjuvant chemotherapy (NAC) and prediction of residual disease in breast cancer using contrast-enhanced spectral mammography (CESM). Forty-two breast cancer patients scheduled for receiving NAC were included. All patients underwent two CESM examinations: pre and post NAC. To assess the response to neoadjuvant chemotherapy, we used a mathematical image analysis software that can calculate the difference in the intensity of enhancement between the pre and post neoadjuvant contrast images (MATLAB and Simulink) (Release 2013b). The proposed technique used the pre and post neoadjuvant contrast images as inputs. The technique consists of three main steps: (1) preprocessing, (2) extracting the region of interest (ROI), and (3) assessment of the response to chemotherapy by measuring the percentage of change in the intensity of enhancement of malignant lesions in the pre and post neoadjuvant CESM studies using a quantitative mathematical technique. This technique depends on the analysis of number of pixels included within the ROI. We compared this technique with the currently used method of evaluation: RECIST 1.1 (response evaluation criteria in solid tumors 1.1) and using another combined response evaluation approach using both RECIST 1.1 in addition to a subjective visual evaluation. Results were then correlated with the postoperative pathology evaluation using Miller–Payne grades. For statistical evaluation, patients were classified into responders and non-responders in all evaluation methods.ResultsAccording to the Miller–Payne criteria, 39/42 (92.9%) of the participants were responders (Miller–Payne grades III, IV, and IV) and 3/42 (7.1%) were non-responders (Miller–Payne grades I and II). Using the proposed technique, 39/39 (100%) were responders in comparison to 38/39 patients (97.4%) using the combined criteria and 34/39 (87.2%) using the RECIST 1.1 evaluation. The calculated correlation coefficient of the proposed quantitative objective mathematical technique, RECIST 1.1 criteria, and the combined method was 0.89, 0.59, and 0.69 respectively. With classification of patients into responder and non-responders, the objective mathematical evaluation showed higher sensitivity, positive and negative predictive values, and overall accuracy (100%, 97.5%, 100%, and 85.7% respectively) compared to RECIST 1.1 evaluation (87.2%, 97.1%, 28.6%, and 54.8% respectively) and the combined response method (97.4%, 97.4%, 66.7%, and 85.7% respectively).ConclusionQuantitative mathematical objective evaluation using CESM images allows objective quantitative and accurate evaluation of the response of breast cancer to chemotherapy and is recommended as an alternative to the subjective techniques as a part of the pre-operative workup.

Post-neoadjuvant treatment and the management of residual disease in breast cancer: state of the art and perspectives.

Achieving a pathologic complete response after neoadjuvant treatment is associated with improved prognosis in breast cancer. The CREATE-X trial demonstrated a significant survival improvement with capecitabine in patients with residual invasive disease after neoadjuvant chemotherapy, and the KATHERINE trial showed a significant benefit of trastuzumab-emtansine (TDM1) in human epidermal growth factor receptor 2 (HER2)-positive patients who did not achieve a pathologic complete response after neoadjuvant treatment, creating interesting alternatives of post-neoadjuvant treatments for high-risk patients. New agents are arising as therapeutic options for metastatic breast cancer such as the cyclin-dependent kinase inhibitors and the immune-checkpoint inhibitors, but none has been incorporated into the post-neoadjuvant setting so far. Evolving techniques such as next-generation sequencing and gene expression profiles have improved our knowledge regarding the biology of residual disease, and also on the mechanisms involved in treatment resistance. The present manuscript reviews the current available strategies, the ongoing trials, the potential biomarker-guided approaches and the perspectives for the post-neoadjuvant treatment and the management of residual disease after neoadjuvant treatment in breast cancer.

Адъювантная цитостатическая терапия минимальной остаточной болезни при раке молочной железы

Адъювантная цитостатическая терапия минимальной остаточной болезни при раке молочной железы

Abstract 591: Clinical profile and outcome associated with breast cancer recurrence after pathologic complete response to neoadjuvant chemotherapy: A multicenter study

Abstract Background: Neoadjuvant chemotherapy (NAC) is a widely accepted treatment not only for locally advanced breast cancer, but also for earlier-stage operable disease. The main purpose of it is to downstage the tumor and to avoid the extensive operation and increase breast conservation surgery. Recent studies have indicated that patients who achieve a pathologic complete response (pCR) after NAC show better outcomes than patients with residual disease in breast cancer (non-pCR). On the other hand, there are some cases in which cancer recurred despite achieving pCR (pCR-rec). This retrospective study investigates the clinical profiles and outcome associated with the patients whose tumor recurred after achieving pCR after NAC. Patients and Methods: All the clinical and pathologic data were collected from 4 institutions in Japan: Yokohama City University Hospital, Kanagawa Cancer Center, Yokohama Rosai Hospital and Tokyo Medical University Hospital. Total of 1,599 operable breast cancer patients who underwent NAC were enrolled. The median follow-up period was 41 months. Vast majority of the patients were treated with anthracycline followed by taxane-based chemotherapy, with or without trastuzumab as neoadjuvant therapy. We defined pCR as no residual invasive cancer in the breast and axially nodes, and non-pCR was divided into 2 groups as Grade 1 (no to mild response) and Grade 2 (moderate response). Results: Of 1,599 patients enrolled in the study, 363 patients achieved pCR (22.7%). Among the pCR patients, 41 were Luminal type, 93 were Luminal-HER2 type, 109 were HER2 type, and 120 were triple-negative (TN) type (pCR rate was 5.94%, 30.4%, 49.3% and 31.3%, respectively). Cancer recurrence after resection was observed in 18 patients (pCR-rec, 4.9%). Axial nodal metastasis diagnosed before NAC associated to cancer recurrence among the pCR patients (p= 0.0003). pCR patients showed significantly better outcome compared to non-pCR, consistent with the previous reports among all patients. However, pCR-rec showed significantly worse DFS compared to those with Grade 1, Grade 2, or pCR (P<0.005 ; 2-year disease-free rate was 33.3%, 79.1%, 88.9% and 98.6%, respectively), as well as HER2 and TN cohort showed consistent results (P<0.005; 2-year disease-free rate was 14.5%, 63.1%, 83.7%,and 95.0%, and P<0.005; 2-year disease-free rate was 17.9%, 55.7%, 81.7% and [no data]). Conclusions: Although achieving pCR after NAC improved outcome, patients with cancer recurrence after pCR show significantly unfavorable outcome even compared to non-pCR patients. In further study, identifying these patients before treatment could improve their poor survival. Citation Format: Mariko Asaoka, Takashi Ishikawa, Eiichi Sato, Kazutaka Narui, Takashi Chishima, Akimitsu Yamada, Nobuyasu Suganuma, Tsutomu Kawaguchi, Kazuaki Takabe. Clinical profile and outcome associated with breast cancer recurrence after pathologic complete response to neoadjuvant chemotherapy: A multicenter study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 591.

Abstract OT2-07-09: Detection and targeting of minimal residual disease in breast cancer to reduce recurrence: The PENN-SURMOUNT and CLEVER trials

Abstract Background: Recurrent breast cancers arise from minimal residual disease (MRD): the pool of disseminated and circulating tumor cells (DTCs and CTCs) that survive in their host following treatment of primary breast cancer. Detection of DTCs in the bone marrow (BM) after treatment is strongly associated with an increased risk of recurrence. Through the analysis of novel genetically-engineered mouse models, we have generated a substantial body of evidence that autophagy and mTOR signaling play key roles in the survival of DTCs. Moreover, administration of agents that block these pathways in mice harboring MRD reduces DTC burden and concomitantly reduces tumor recurrence, providing the rationale for translating these findings to patients (pts). Trial Design: The PENN-SURMOUNT screening study uses a clinically validated IHC assay (DTC-IHC) to identify at-risk pts who harbor DTCs. DTC+ pts are eligible for enrollment on the CLEVER trial, which will determine the feasibility, safety and efficacy of administering hydroxychloroquine (HCQ) and/or everolimus (EVE) in DTC+ patients to target MRD and prevent recurrence. PENN-SURMOUNT is single center, prospective cohort study of pts who have completed therapy for primary breast cancer, are within 5 yrs of diagnosis and are at increased risk for relapse by virtue of nodal positivity, triple negative disease, ER+/Oncotype DX RS ≥ 25, or residual disease after neoadjuvant therapy. Pts undergo screening BM aspirate to test for DTCs following completion of adjuvant chemo and radiotherapy. The primary objective of the study is to determine the incidence and frequency of MRD in pts who have completed primary treatment for breast cancer and to ascertain eligibility for the CLEVER recurrence prevention trial. CLEVER is a randomized, controlled, open label phase II pilot trial. Target enrollment is 60 pts, with 15 pts allocated to each of 4 treatment arms: HCQ (600 mg BID), EVE (10mg daily), combination HCQ/EVE, or control/observation. A cycle is 28 days of continuous dosing. After a 3-month observation period, control pts will be offered HCQ/EVE therapy for 6 cycles; thus, the control group is actually a delayed treatment group and all pts will receive treatment. Pts who demonstrate persistent DTCs after 6 cycles will continue on combination therapy for an additional 6 cycles. The primary endpoint is feasibility of administering HCQ, EVE or the combination in this population. Secondary objectives include safety, efficacy (DTC reduction), and 3-year RFS. The principal translational objective is to assess the utility of a novel DTC assay, "DTC-Flow", for more sensitive detection and response to study therapy, compared to DTC-IHC. Additional translational objectives include determining whether patient DTCs, CTCs, and cell-free circulating plasma tumor DNA (ptDNA) biologically reflect the primary tumor and predict response. As of 5/23/17, 58 patients have been enrolled to PENN SURMOUNT, with a DTC-positivity rate of 22.6%; CLEVER opened in 2/2017; 11 patients are currently enrolled. Contact information: angela.demichele@uphs.upenn.edu Key words: Recurrence, disseminated tumor cells, dormancy, minimal residual disease, autophagy, mTOR, Everolimus, hydroxychloroquine Citation Format: Bayne LJ, Nivar I, Goodspeed B, Wileyto P, Savage J, Shih NNC, Feldman MD, Edwards J, Clark AS, Fox KR, Matro JM, Domchek SM, Bradbury AR, Shah PD, Chislock EM, Belka GK, Wang J, Amaravadi R, Chodosh LA, DeMichele AM. Detection and targeting of minimal residual disease in breast cancer to reduce recurrence: The PENN-SURMOUNT and CLEVER trials [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-07-09.

Minimal residual disease in breast cancer: an overview of circulating and disseminated tumour cells.

Within the field of cancer research, focus on the study of minimal residual disease (MRD) in the context of carcinoma has grown exponentially over the past several years. MRD encompasses circulating tumour cells (CTCs)—cancer cells on the move via the circulatory or lymphatic system, disseminated tumour cells (DTCs)—cancer cells which have escaped into a distant site (most studies have focused on bone marrow), and resistant cancer cells surviving therapy—be they local or distant, all of which may ultimately give rise to local relapse or overt metastasis. Initial studies simply recorded the presence and number of CTCs and DTCs; however recent advances are allowing assessment of the relationship between their persistence, patient prognosis and the biological properties of MRD, leading to a better understanding of the metastatic process. Technological developments for the isolation and analysis of circulating and disseminated tumour cells continue to emerge, creating new opportunities to monitor disease progression and perhaps alter disease outcome. This review outlines our knowledge to date on both measurement and categorisation of MRD in the form of CTCs and DTCs with respect to how this relates to cancer outcomes, and the hurdles and future of research into both CTCs and DTCs.

Abstract P5-04-06: Analysis of STEAP1 expression as a therapeutic target in breast cancer

Abstract Objective: The six transmembrane epithelial antigen of the prostate (STEAP1) is predominantly overexpressed in human prostate cancer. STEAP1 was first identified as a prostate-specific cell-surface antigen and found to be up-regulated in various cancers including lung, bladder, colon, and ovarian with little expression in normal tissue. An anti-STEAP1 monoclonal antibody linked to an antimitotic agent is currently in Phase I clinical trials for prostate cancer patients. Microarray data from our lab suggested that STEAP1 is also highly expressed in human breast cancers and bone marrow disseminated tumor cells. In this study we evaluate expression STEAP1 in primary tumors, and bone marrow (BM) from breast cancer patients. Experimental procedures: RNA was isolated from primary tumor, non-malignant breast tissue and bone marrow (BM) from stage II and III breast cancer patients, healthy volunteers, breast cancer cell lines and BM from patient derived xenographs (PDX). Disseminated tumor cells (DTCs) from patient BM were enriched by microfiltration and analyzed by RNA-in situ hybridization (ISH). STEAP1 RNA expression was analyzed by Nanostring nCounter and qRT-PCR using human specific probes. STEAP1 immunohistochemical (IHC) staining of human tissue was performed using standard protocols. Knockdown of steap1 expression was accomplished using a lentiviral system. Results: STEAP1 mRNA was up-regulated in 77% of tumors (28/36) compared to the corresponding normal tissue. STEAP1 protein was expressed in 100% of tumors (8/8) and was absent in non-malignant breast tissue (7/7) by IHC staining. STEAP1 mRNA was not expressed normal BM, but was detected in 8% (6/74) of BM from patients with early stage breast cancer. STEAP1 expression in the BM was associated with triple negative disease (3/6) and recurrent disease development (4/6, p=.028). STEAP1 expression was observed in individual DTCs isolated from patients BM, while no expression was observed in normal BM. In a PDX model of breast cancer, STEAP1 expression in BM was only observed in mouse who developed metastatic disease associated (7/10, p=.004). Knockdown of STEAP1 in the breast cancer cell line MDA-MB231 cells inhibited cell growth by 80-90%. Conclusion: STEAP1 is expressed in human breast tumors and disseminated tumor cells found in the bone marrow of breast cancer patients. Expression of STEAP1 in the BM is significantly associated with the development of metastatic disease in patients as well as in a mouse model of breast cancer. Our data indicate that STEAP1 could serve as a therapeutic target for the treatment of minimal residual disease in breast cancer. Citation Format: Aft R, Mudalagiriyappa C, Pillai S, Watson M. Analysis of STEAP1 expression as a therapeutic target in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-06.

Abstract 5258: Expression analysis of STEAP1 in breast cancer patients as therapeutic target

Abstract Objective: The six transmembrane epithelial antigen of the prostate (STEAP1) is predominantly overexpressed in human prostate cancer. STEAP1 was first identified as a prostate-specific cell-surface antigen and found to be up-regulated in multiple cancer cell lines and in various cancers, including lung, bladder, colon and ovarian cancers. However no significant expression in normal tissue has been reported suggesting its potential use as a target for anti-STEAP1 immunotherapy. An anti-STEAP1 monoclonal antibody linked to an antimitotic agent is in Phase I clinical trials for prostate cancer patients. Microarray data from our lab suggested that STEAP1 is also highly expressed in human breast cancers. In this study we evaluate expression STEAP1 in primary tumors and the bone marrow from breast cancer patients as a therapeutic target for minimal residual disease. Experimental procedures: RNA was isolated from primary tumor and non-malignant breast tissue. RNA was also isolated from bone marrow of state II and III breast cancer patients, healthy volunteers and breast cancer cell lines. STEAP1 expression was analyzed by nanostring nCounter and real time qRT-PCR using human specific probes. STEAP1 immunohistochemical (IHC) staining of human tissue was performed using standard IHC Protocol. Results: STEAP1 mRNA was up-regulated in 77% of tumors (28/36) compared to the corresponding normal tissue. STEAP1 protein was expressed in 100% of tumors (8/8) and was absent in non-malignant breast tissue (7/7) by IHC staining. STEAP1 mRNA was not expressed normal BM, but was detected in 8% (6/74) of BM from patients with early stage breast cancer. Of patients with STEAP1 positive BM, 50% (3/6) had triple negative disease and 66% (4/6) developed recurrent disease (p = .028). Conclusion: STEAP1 is expressed in human breast tumors and the BM of breast cancer patients. Expression of STEAP1 in the BM is significantly associated with the development of metastatic disease. Our data indicate that STEAP1 could serve as a therapeutic target for the treatment of minimal residual disease in breast cancer. Citation Format: Chidananda Mudalagiriyappa, Sreeraj Pillai, Mark Watson, Rebecca Aft. Expression analysis of STEAP1 in breast cancer patients as therapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5258. doi:10.1158/1538-7445.AM2015-5258

Minimal Residual Disease in Breast Cancer: In Blood Veritas

A blood-based molecular test might direct recommendations for systemic therapies in patients with early-stage breast cancer undergoing surgery with curative intent. A new study suggests that droplet digital PCR (ddPCR) can be used to detect cancer-specific DNA alterations in plasma with sensitivity suitable for monitoring minimal residual disease.

Does magnetic resonance imaging accurately predict residual disease in breast cancer?

Background The accuracy of magnetic resonance imaging (MRI) in identifying residual disease after breast conservation therapy (BCT) is unclear. Method Review of an institutional database identified patients with positive or close (≤2 mm) margins undergoing MRI before re-excision. Histopathologic correlation was performed. Results Forty-three women underwent MRI after BCT. MRI suggested residual disease in 29 patients, of whom 20 (69%) had residual carcinoma pathologically. Nine patients had false-positive MRI as seen by benign pathology findings. Fourteen MRIs indicated no residual disease, of which 6 had residual disease pathologically. The sensitivity and positive predictive value of MRI was 77% and 69%, respectively. MRI conducted within 28 days of the original surgery was 85% sensitive. MRI performed after 28 days was 69% sensitive. Conclusions MRI is able to detect residual disease among most patients undergoing re-excision. False-positive results may be caused by inflammatory processes that resemble residual disease.

Prognostic relevance of the AQP5 −1364C>A polymorphism in primary breast cancer

Besides known prognostic factors in breast cancer, disseminated tumor cells are regarded as a surrogate marker for minimal residual disease in breast cancer. Their prognostic significance is comparable to that of lymph node status. However, the mechanism by which the primary tumor directs cells to disseminate into lymph nodes or into blood vessels is unclear. Aquaporins (AQPs) are small integral membrane proteins that provide a major pathway for water transport throughout several organs, and have been shown to be of potential importance in various types of cancer. Here, we analyzed the single nucleotide polymorphism (SNP) A(-1364)C in AQP5 in 107 patients with early stage breast cancer in order to test the hypothesis that this polymorphism is associated with lymphogenous and hematogenous tumor cell dissemination and overall survival. Paraffin-embedded tumor tissue was dewaxed, and DNA was extracted from tumor tissues using the QIAamp Blood DNA Mini Kit. The quantification and quality of the extracted DNA were determined spectrophotometrically. Genotyping of the -1364A>C polymorphism was performed by Pyrosequencing. Cytokeratin-positive (CK+) bone marrow (BM) cells were isolated by density gradient centrifugation followed by immunocytochemistry, applying the pan CK antibody A45-B/B3. There was no evidence of an association between the AQP5C>A genotypes and an increased risk of developing breast cancer, nor between the genotypes and tumor size, lymph node involvement, distant metastasis, grade, histopathology, expression of estrogen receptor and HER2, or the dissemination of tumor cells to BM. In contrast, when comparing C-allele carriers with patients carrying the AA genotype, expression of the progesterone receptor differed significantly between these genotype groups (mean AA genotype 51%, mean AC and CC genotype 73%; p=0.039). Additionally, a significant correlation was found between progesterone receptor expression and adjuvant chemotherapy (p=0.021) and adjuvant endocrine therapy (p=0.017), respectively. SNPs in AQP5 are not associated with hematogenous or lymphogenous tumor cell spread. However, we observed for the first time an association between SNPs in AQP5 and progesterone receptor positivity, which might have implications for future adjuvant treatment. Further investigations must include more than one AQP as well as factors promoting angiogenesis to elucidate the different modes of tumor cell dissemination.