Cristina I. Truica

Acoustic separation of circulating tumor cells

Significance The separation and analysis of circulating tumor cells (CTCs) provides physicians a minimally invasive way to monitor the response of cancer patients to various treatments. Among the existing cell-separation methods, acoustic-based approaches provide significant potential to preserve the phenotypic and genotypic characteristics of sorted cells, owing to their safe, label-free, and contactless nature. In this work, we report the development of an acoustic-based device that successfully demonstrates the isolation of rare CTCs from the clinical blood samples of cancer patients. Our work thus provides a unique means to obtain viable and undamaged CTCs, which can subsequently be cultured. The results presented here offer unique pathways for better cancer diagnosis, prognosis, therapy monitoring, and metastasis research. Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.

Flexible Micro Spring Array Device for High-Throughput Enrichment of Viable Circulating Tumor Cells

BACKGROUND The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression. METHODS Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients. RESULTS The FMSA device enriched tumor cells with 90% capture efficiency, higher than 10(4) enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm(2) device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples. CONCLUSIONS The FMSA device provides a versatile platform capable of viable enrichment and analysis of CTCs from clinically relevant volumes of whole blood.

Neoadjuvant Concurrent Paclitaxel and Radiation in Stage II/III Breast Cancer

Purpose: The aim of this study was to determine the safety and pathologic response rates following neoadjuvant paclitaxel and radiation in patients with stage II/III breast cancer and to evaluate the use of sequential biopsies to allow an in vivo assessment of biological markers as potential predictive markers of response to this regimen. Patients and Methods: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel 175 mg/m2 every 3 weeks, followed by twice-weekly paclitaxel 30 mg/m2 and concurrent radiation. Core biopsies were obtained at baseline and 24 to 72 hours after the first cycle of paclitaxel. After completing neoadjuvant treatment, patients underwent definitive surgery. The primary end point was pathologic complete response, which is defined as the absence of any invasive cancer at surgery. Potential markers of therapeutic response were evaluated including markers of proliferation, apoptosis, p21, HER2, estrogen receptor, and progesterone receptor status. Results: Of the 38 patients enrolled, 13 (34%) had a pathologic complete response. There was no significant difference in baseline Ki-67 between responders (35%) and nonresponders (28%; P = 0.45). There was also no significant change in Ki-67 following paclitaxel administration. Despite this lack of immunohistologic change in proliferative activity, baseline mitotic index was higher for patients with pathologic complete response over nonresponders (27 versus 10, P = 0.003). Moreover, the increase in mitotic index following paclitaxel administration was associated with pathologic complete response. Conclusions: Neoadjuvant paclitaxel/radiation is effective and well tolerated. Tumor proliferation at baseline and response to chemotherapy as measured by mitotic activity may serve as an important indicator of pathologic response to neoadjuvant paclitaxel/radiation.

Abstract S1-02: PrECOG 0102: A randomized, double-blind, phase II trial of fulvestrant plus everolimus or placebo in post-menopausal women with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC) resistant to aromatase inhibitor (AI) therapy

Background: Although AIs are an effective treatment for HR-positive MBC, whether used alone or in combination with CDK4/6 inhibitors, resistance to therapy and disease progression invariably develops. Therapeutic options for AI-resistant disease include the m-TOR inhibitor everolimus in combination with the steroidal AI exemestane, or the selective estrogen receptor downregulator (SERD) fulvestrant alone. We hypothesized that the combination of fulvestrant and everolimus would be more effective than fulvestrant alone in AI-resistant MBC. Methods: Major eligibility criteria included post-menopausal women with HR-positive, HER2-negative MBC, recurrence or progression while receiving prior non-steroidal or steroidal AI therapy, ECOG PS 0-1, and ≤1 prior chemotherapy regimen for metastases. All patients received fulvestrant (500mg IM q2 weeks for 3 doses, then q4 weeks) plus oral everolimus (10mg) or placebo (1:1 randomization). Prophylactic corticosteroid mouthwash was not used. Tumor assessment was performed at baseline and every 12 weeks. Treatment continued until progressive disease (PD) by RECIST 1.1 criteria. Patients who discontinued everolimus/placebo due to toxicity continued fulvestrant until PD. The primary endpoint was progression-free survival (PFS), defined as time from start of treatment until progression or death. With accrual of 130 patients (120 eligible), the trial had 90% power to detect an improvement in median PFS from 5.4 months to 9.2 months (1-sided stratified log-rank test, type I error rate10%), with analysis planned after 98 PFS events. Results: Of 130 patients treated (64 everolimus, 66 placebo), median age was 61 years (range 35-92), and treatment arms were balanced for stratification factors used in randomization, including ECOG PS 0 vs. 1 (59%/41%), measurable disease (66%), and prior chemotherapy for metastasis (17%). Grade 3/4 AEs were more common in the everolimus arm (53%/3% vs. 23%3%), including hyperglycemia (16%/0% vs. 0%), stomatitis (11%/0% vs. 0%), hypertriglyceridemia (9%/2% vs. 0%), lymphopenia (9%/0% vs. 0%), and pneumonitis (6%/2% vs. 0%). There were 3 grade 5 events (2 everolimus, 1 placebo arm), none of which were attributed to therapy. Selected grade 2 events of interest included fatigue (17% vs. 6%), hyperglycemia (6% vs. 0%), and stomatitis (6% vs. 0%). Everolimus/placebo was discontinued for adverse events, patient withdrawal, or physician discretion in 39% in the everolimus arm and 21% in the placebo arm. After 98 PFS events, median PFS was 10.4 months in the everolimus arm versus 5.1 months in the placebo arm (hazard ratio: 0.61, 95% C.I. 0.40 – 0.92; stratified logrank p= 0.02). Conclusions: The addition of everolimus to fulvestrant significantly improved PFS in post-menopausal women with AI-resistant MBC. Everolimus used without prophylactic corticosteroid mouthwash exhibited a similar rate of oral mucositis and overall AE profile when combined with fulvestrant as when combined with exemestane. Keywords: advanced breast cancer, PrECOG 0102, endocrine resistance, everolimus, exemestane, hormone receptor-positive, mTOR inhibitor, postmenopausal. Citation Format: Kornblum NS, Manola J, Klein P, Ramaswamy B, Brufsky A, Stella PJ, Burnette B, Telli M, Makower DF, Leach J, Truica CI, Wolff AC, Soori GS, Haley B, Nagarajan A, Wassenaar TR, Goldstein L, Miller KD, Sparano JA. PrECOG 0102: A randomized, double-blind, phase II trial of fulvestrant plus everolimus or placebo in post-menopausal women with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC) resistant to aromatase inhibitor (AI) therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-02.

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.

A multiplexed marker-based algorithm for diagnosis of carcinoma of unknown primary using circulating tumor cells

Real-time, single-cell multiplex immunophenotyping of circulating tumor cells (CTCs) is hypothesized to inform diagnosis of tissue of origin in patients with carcinoma of unknown primary (CUP). In 20 to 50% of CUP patients, the primary site remains unidentified, presenting a challenge for clinicians in diagnosis and treatment. We developed a post-CellSearch CTC assay using multiplexed Q-dot or DyLight conjugated antibodies with the goal of detecting multiple markers in single cells within a CTC population. We adapted our approach to size-based CTC enrichment protocols for capturing CTCs and subsequent immunofluorescence (IF) using a minimal set of markers to predict the primary sites for common metastatic tumors. The carcinomas are characterized with cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA. IF has been optimized in cultured tumor cells with individual antibodies, then with conjugated antibodies to form a multiplex antibody set. With IF, we evaluated antibodies specific to these 5 markers in lung, breast, colorectal, and prostate cancer cell lines and blood from metastatic prostate and breast cancer patients. This advanced technology provides a noninvasive, diagnostic blood test as an adjunct to routine tissue biopsy. Its further implementation requires prospective clinical testing.

Metastatic Prostatic Adenocarcinoma Mimicking Inflammatory Breast Carcinoma: A Case Report

Prostate adenocarcinoma can manifest as a fairly indolent tumor or as a very aggressive cancer with significant invasive and metastatic potential. Common metastatic sites include bone, liver, lymph nodes, and adrenal glands. Dermatologic manifestations are rare. We present a case of a man who presented with breast skin changes that mimicked inflammatory breast carcinoma with specialized testing ultimately giving a diagnosis of metastatic prostatic adenocarcinoma. A 78-year-old man presented with left breast redness and swelling. Examination revealed an erythematous rash with subcutaneous edema over the left hemithoracic area. A breast ultrasound showed no focal mass, and a breast core biopsy had no evidence of tumor. A skin biopsy showed metastatic carcinoma in dermal lymphatics, and the tumor was found to have no estrogen or progesterone receptors or HER2 expression. Computed tomography scans, positron emission tomography, and a nuclear bone scan revealed widespread skeletal metastases. The patient received a 3-month course of capecitabine and cyclophosphamide with no improvement in his skin lesions. Subsequent immunohistochemical staining on the tumor specimen was positive for prostate-specific antigen (PSA) and alpha-methyl-CoA-racemase, confirming a diagnosis of metastatic prostatic adenocarcinoma. He received leuprolide and bicalutamide and demonstrated significant improvement with near-complete resolution of his skin lesions and a decrease in his PSA level. Prostatic adenocarcinoma presenting initially as a breast malignancy is a rarely recognizable clinical event. Undoubtedly, increased awareness and recognition of the rare entity described herein will allow for the prompt initiation of specific therapies, which might be of benefit to many patients.

An Analysis of Oncotype DX Recurrence Scores and Clinicopathologic Characteristics in Invasive Lobular Breast Cancer

The Oncotype DX breast cancer assay (Genomic Health, Redwood City, CA) is increasingly being used to guide treatment decisions for patients with early stage, hormone‐positive, Her‐2‐negative breast cancer. The utility of the Oncotype DX in decision making for treatment of invasive lobular carcinoma (ILC) has not been investigated as the results reported by Genomic Health are largely in a population with invasive ductal carcinoma (IDC). The authors hypothesized that the Oncotype DX recurrence score (RS) distribution for ILC is different than that for IDC. We performed a retrospective analysis of early stage breast cancer patients treated at Penn State Cancer Institute from 2001 to 2011 and identified 102 patients with ILC. We also pulled RS data from our institutions prospective registry of consecutive patients with early stage IDC treated during the same time period. Median follow‐up was 55 months. We found that the RS distribution for ILC differed significantly from that of IDC (p = 0.024). We also found a statistically significant difference in the RS distribution between the pure ILC and pleomorphic ILC subtypes (p = 0.027). The Oncotype DX RS distribution in ILC is unique, differing significantly from that in ductal carcinoma. Consequently, the clinical usefulness and cost‐effectiveness of the Oncotype DX in guiding treatment for ILC should be further investigated.

Randomized Phase II Trial of Fulvestrant Plus Everolimus or Placebo in Postmenopausal Women With Hormone Receptor–Positive, Human Epidermal Growth Factor Receptor 2–Negative Metastatic Breast Cancer Resistant to Aromatase Inhibitor Therapy: Results of PrE0102

Purpose The mammalian target of rapamycin inhibitor everolimus targets aberrant signaling through the PI3K/AKT/mammalian target of rapamycin pathway, a mechanism of resistance to anti-estrogen therapy in estrogen receptor (ER)-positive breast cancer. We hypothesized that everolimus plus the selective ER downregulator fulvestrant would be more efficacious than fulvestrant alone in ER-positive metastatic breast cancer resistant to aromatase inhibitor (AI) therapy. Patients and Methods This randomized, double-blind, placebo-controlled, phase II study included 131 postmenopausal women with ER-positive, human epidermal growth factor receptor 2-negative, AI-resistant metastatic breast cancer randomly assigned to fulvestrant (500 mg days 1 and 15 of cycle 1, then day 1 of cycles 2 and beyond) plus everolimus or placebo. The study was designed to have 90% power to detect a 70% improvement in median progression-free survival from 5.4 months to 9.2 months. Secondary end points included objective response and clinical benefit rate (response or stable disease for at least 24 weeks). Prophylactic corticosteroid mouth rinses were not used. Results The addition of everolimus to fulvestrant improved the median progression-free survival from 5.1 to 10.3 months (hazard ratio, 0.61 [95% CI, 0.40 to 0.92]; stratified log-rank P = .02), indicating that the primary trial end point was met. Objective response rates were similar (18.2% v 12.3%; P = .47), but the clinical benefit rate was significantly higher in the everolimus arm (63.6% v 41.5%; P = .01). Adverse events of all grades occurred more often in the everolimus arm, including oral mucositis (53% v 12%), fatigue (42% v 22%), rash (38% v 5%), anemia (31% v. 6%), diarrhea (23% v 8%), hyperglycemia (19% v 5%), hypertriglyceridemia (17% v 3%), and pneumonitis (17% v 0%), although grade 3 to 4 events were uncommon. Conclusion Everolimus enhances the efficacy of fulvestrant in AI-resistant, ER-positive metastatic breast cancer.

Antibody Therapy for Solid Tumors

Over the last several years the use of monoclonal antibody (MAb) therapy for the treatment of human malignancy has advanced from experimental therapy to standard of care for some malignancies. The success of MAb therapy stems not only from the ability to humanize the antibody, thus, decreasing the inherent immunogenicity of the approach but also from targeting appropriate proteins. Human cancers associated with the overexpression of particular growth factor receptors such as HER-2/neu, epidermal growth factor receptor, and vascular endothelial growth factor, have been impacted by commercially available antibodies. This review focuses on the mechanisms of action and clinical benefit of MAb therapy against common growth factor receptors.