KRAS G12C Inhibitors in the Treatment of Metastatic Colorectal Cancer.

KRAS mutations contribute substantially to the overall colorectal cancer burden and have long been a focus of drug development efforts. After a lengthy preclinical road, KRAS inhibition via the G12C allele has finally become a therapeutic reality. Unlike in NSCLC, early studies of KRAS inhibitors in CRC struggled to demonstrate single agent activity. Investigation into these tissue-specific treatment differences has led to a deeper understanding of the complexities of MAPK signaling and the diverse adaptive feedback responses to KRAS inhibition. EGFR reactivation has emerged as a principal resistance mechanism to KRAS inhibitor monotherapy. Thus, the field has pivoted to dual EGFR/KRAS blockade with promising efficacy. Despite significant strides in the treatment of KRAS G12C mutated CRC, new challenges are on the horizon. Alternative RTK reactivation and countless acquired molecular resistance mechanisms have shifted the treatment goalpost. This review focuses on the historical and contemporary clinical strategies of targeting KRAS G12C alterations in CRC and highlights future directions to overcome treatment challenges.

Rare malignant glomus tumor of the esophagus with pulmonary metastasis: a case report.

Background: Glomus tumors are typically benign soft tissue neoplasms that arise in peripheral cutaneous structures. Visceral organ involvement is exceedingly rare. Case Description: Here we present a case of malignant glomus tumor of the esophagus with pulmonary metastases in a 57-year-old woman presenting with three weeks of progressive dysphagia, epigastric pain, and 35-pound weight loss. Upper endoscopy revealed a 5×3.5×2.5 cm vascular esophageal mass. Contrast-enhanced CT showed multiple, scattered sub-centimeter pulmonary nodules bilaterally. Diagnosis of metastatic glomus tumor was confirmed immunohistochemically on primary tumor and lung biopsies. Localized resection was not feasible due to the patient's poor condition. A trial of gemcitabine and docetaxel was planned, but the patient experienced rapid clinical deterioration after a single dose of gemcitabine before electing for hospice care. Conclusions: We have reviewed the 11 other published cases of esophageal glomus tumors, only one of which was similarly metastatic at time of presentation. Of those patients with localized disease treated with surgical excision, all were alive and had no evidence of recurrence (NER) at their times of publication. In contrast, disease ultimately progressed despite surgery and chemoradiotherapy in the sole other case of metastatic glomus tumor of the esophagus. Although glomus tumors are largely benign entities, this case highlights their rare and aggressive malignant potential.

Evaluation of Dose Distribution to Organs-at-Risk in a Prospective Phase 1 Trial of Pembrolizumab and Multisite Stereotactic Body Radiation Therapy (SBRT).

PURPOSE: Our purpose was to characterize the radiation doses to organs-at-risk (OAR) in the phase I trial (NCT02608385) that established safety/efficacy of stereotactic body radiation therapy (SBRT) using NRG-BR001 dose constraints combined with programmed cell death protein 1 blockade for metastatic disease. METHODS AND MATERIALS: Between January 2016 and May 2018, 73 patients with advanced solid tumors were treated with SBRT followed by pembrolizumab. Tumor volumes (gross tumor volume/internal tumor volume) were delineated for each metastasis, with planning target volume contraction to limit OAR dose per protocol (n = 54) or when gross tumor volume/internal tumor volume > 65 cm3 (n = 19). For 20 OAR, doses were compared with NRG-BR001 constraints. Protocol constraints were considered challenged when the minimum of the highest dose received by ≥6 patients without dose-limiting toxicities (DLTs) (Dmax6th) was ≥70% of the protocol constraint. RESULTS: A total of 151 metastases were irradiated including 32 peripheral lung, 23 central lung, 13 mediastinal/cervical, 24 liver, 28 abdominal-pelvic, 16 osseous, and 15 spinal metastases. A median of 2 metastases (range, 2-4) with mean volumes of 33.5 cm3 (range, 0.4-391 cm3) were treated using average planning target volumes of 50.7 cm3 (range, 3.2-161 cm3). At least 1 dose constraint from NRG-BR001 was exceeded in 38 of 73 (52%) patients. OAR constraints were challenged in 10 serial organs (gastrointestinal, cardio-pulmonary, musculoskeletal, and nervous systems) and 1 parallel OAR (lung). Grade 3 DLTs occurred in 6 patients, including pneumonitis (n = 3), colitis (n = 2), and hepatic failure (n = 1). In 4 patients, the toxicity could be directly attributed to the planned dose to OAR (ie, pneumonitis due to high lung dose or colitis due to high bowel dose). CONCLUSIONS: Multisite SBRT in combination with programmed cell death protein 1 blockade was safely tolerated when treating critical central, abdominal-pelvic, and peripheral OAR nearing NRG-BR001 constraints with clinically acceptable toxicity in the corresponding organ systems. The observed relationship between dose and DLTs in 4 of 6 patients indicates that NRG-BR001 dose constraints should be respected in subsequent trials to maintain clinical safety.

A study of the dosimetric impact of daily setup variations measured with cone-beam CT on three-dimensional conformal radiotherapy for early-stage breast cancer delivered in the prone position.

PURPOSE: To evaluate the dosimetric impact of daily positioning variations measured with cone-beam computed tomography (CBCT) on whole-breast radiotherapy patients treated in the prone position. METHODS: Daily CBCT was prospectively acquired for 30 consecutive patients positioned prone. Treatment for early-stage (≤II) breast cancer was prescribed with standard dose (50 Gy/25 fractions) or hypofractionation (42.56 Gy/16 fractions) for 13 and 17 patients, respectively. Systematic and random errors were calculated from the translational CBCT shifts and used to determine population-based setup margins. Mean translations (±one standard deviation) for each patient were used to simulate the dosimetric impact on targets (PTV_eval and lumpectomy cavity), heart, and lung. Paired Student's t tests at α = 0.01 were used to compare dose metrics after correction for multiple testing (P < 0.002). Significant correlation coefficients were used to identify associations (P < 0.01). RESULTS: Of 597 total fractions, 20 ± 13% required patient rotation. Mean translations were 0.29 ± 0.27 cm, 0.41 ± 0.34 cm, and 0.48 ± 0.33 cm in the anterior-posterior, superior-inferior, and lateral directions leading to calculated setup margins of 0.63, 0.88, and 1.10 cm, respectively. Average three-dimensional (3D) shifts correlated with the maximum distance of breast tissue from the sternum (r = 0.62) but not with body-mass index. Simulated shifts showed significant, but minor, changes in dose metrics for PTV_eval, lung, and heart. For left-sided treatments (n = 18), mean heart dose increased from 109 ± 75 cGy to 148 ± 115 cGy. Shifts from the original plan caused PTV_eval hotspots (V105%) to increase by 5.2% ± 3.8%, which correlated with the total MU of wedged fields (r = 0.59). No significant change in V95% to the cavity was found. CONCLUSIONS: Large translational variations that occur when positioning prone breast patients had small but significant dosimetric effects on 3DCRT plans. Daily CBCT may still be necessary to correct for rotational variations that occur in 20% of treatments. To maintain planned dose metrics, unintended beam shifts toward the heart and the contribution of wedged fields should be minimized.

BET inhibition increases ßIII-tubulin expression and sensitizes metastatic breast cancer in the brain to vinorelbine.

Metastases from primary breast cancer result in poor survival. ßIII-tubulin (TUBB3) has been established as a therapeutic target for breast cancer metastases specifically to the brain. In this study, we conducted a systematic analysis to determine the regulation of TUBB3 expression in breast cancer metastases to the brain and strategically target these metastases using vinorelbine (VRB), a drug approved by the U.S. Food and Drug Administration (FDA). We found that human epidermal growth factor receptor 2 (HER2) signaling regulates TUBB3 expression in both trastuzumab-sensitive and trastuzumab-resistant neoplastic cells. We further discovered that bromodomain and extra-terminal domain (BET) inhibition increases TUBB3 expression, rendering neoplastic cells more susceptible to apoptosis by VRB. Orthotopic xenograft assays using two different breast cancer cell models revealed a reduction in tumor volume with BET inhibition and VRB treatment. In addition, in vivo studies using a model of multiple brain metastasis (BM) showed improved survival with the combination of radiation + BET inhibitor (iBET-762) + VRB (75% long-term survivors, P < 0.05). Using in silico analysis and BET inhibition, we found that the transcription factor myeloid zinc finger-1 (MZF-1) protein binds to the TUBB3 promoter. BET inhibition decreases MZF-1 expression and subsequently increases TUBB3 expression. Overexpression of MZF-1 decreases TUBB3 expression and reduces BM in vivo, whereas its knockdown increases TUBB3 expression in breast cancer cells. In summary, this study demonstrates a regulatory mechanism of TUBB3 and provides support for an application of BET inhibition to sensitize breast cancer metastases to VRB-mediated therapy.

Engineered Fibrillar Fibronectin Networks as Three-Dimensional Tissue Scaffolds.

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre-coatings in an attempt to create a cell-supporting environment in both two- and three-dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM-constituting fibrillar protein networks naturally deposited by cells. Here, the cell-free and scalable synthesis of freely suspended and mechanically robust three-dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three-phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial-to-mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre-clinical diagnostics.

FABP7 is a key metabolic regulator in HER2+ breast cancer brain metastasis.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer patients is associated with increased incidence of breast cancer brain metastases (BCBM), but the mechanisms underlying this phenomenon remain unclear. Here, to identify brain-predominant genes critical for the establishment of BCBM, we conducted an in silico screening analysis and identified that increased levels of fatty acid-binding protein 7 (FABP7) correlate with a lower survival and higher incidence of brain metastases in breast cancer patients. We validated these findings using HER2+ BCBM cells compared with parental breast cancer cells. Importantly, through knockdown and overexpression assays, we characterized the role of FABP7 in the BCBM process in vitro and in vivo. Our results uncover a key role of FABP7 in metabolic reprogramming of HER2 + breast cancer cells, supporting a glycolytic phenotype and storage of lipid droplets that enable their adaptation and survival in the brain microenvironment. In addition, FABP7 is shown to be required for upregulation of key metastatic genes and pathways, such as integrins-Src and VEGFA, and for the growth of HER2+ breast cancer cells in the brain microenvironment in vivo. Together, our results support FABP7 as a potential target for the treatment of HER2+ BCBM.

Single-institution report of setup margins of voluntary deep-inspiration breath-hold (DIBH) whole breast radiotherapy implemented with real-time surface imaging.

PURPOSE: We calculated setup margins for whole breast radiotherapy during voluntary deep-inspiration breath-hold (vDIBH) using real-time surface imaging (SI). METHODS AND MATERIALS: Patients (n = 58) with a 27-to-31 split between right- and left-sided cancers were analyzed. Treatment beams were gated using AlignRT by registering the whole breast region-of-interest to the surface generated from the simulation CT scan. AlignRT recorded (three-dimensional) 3D displacements and the beam-on-state every 0.3 s. Means and standard deviations of the displacements during vDIBH for each fraction were used to calculate setup margins. Intra-DIBH stability and the intrafraction reproducibility were estimated from the medians of the 5th to 95th percentile range of the translations in each breath-hold and fraction, respectively. RESULTS: A total of 7269 breath-holds were detected over 1305 fractions in which a median dose of 200 cGy was delivered. Each fraction was monitored for 5.95 ± 2.44 min. Calculated setup margins were 4.8 mm (A/P), 4.9 mm (S/I), and 6.4 mm (L/R). The intra-DIBH stability and the intrafraction reproducibility were ≤0.7 mm and ≤2.2 mm, respectively. The isotropic margin according to SI (9.2 mm) was comparable to other institutions' calculations that relied on x-ray imaging and/or spirometry for patients with left-sided cancer (9.8-11.0 mm). Likewise, intra-DIBH variability and intrafraction reproducibility of breast surface measured with SI agreed with spirometry-based positioning to within 1.2 and 0.36 mm, respectively. CONCLUSIONS: We demonstrated that intra-DIBH variability, intrafraction reproducibility, and setup margins are similar to those reported by peer studies who utilized spirometry-based positioning.

Functional Isolation of Tumor-Initiating Cells using Microfluidic-Based Migration Identifies Phosphatidylserine Decarboxylase as a Key Regulator.

Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses. Using this device, we isolated a small subpopulation of migratory cells with significantly greater tumor formation and metastasis in mouse models. Whole transcriptome sequencing of migratory versus non-migratory cells from two metastatic breast cancer cell lines revealed a unique set of genes as key regulators of tumor-initiating cells. We focused on phosphatidylserine decarboxylase (PISD), a gene downregulated by 8-fold in migratory cells. Breast cancer cells overexpressing PISD exhibited reduced tumor-initiating potential in a high-throughput microfluidic mammosphere device and mouse xenograft model. PISD regulated multiple aspects of mitochondria, highlighting mitochondrial functions as therapeutic targets against cancer stem cells. This research establishes not only a novel microfluidic technology for functional isolation of tumor-initiating cells regardless of cancer type, but also a new approach to identify essential regulators of these cells as targets for drug development.

Imaging Sensitivity of Quiescent Cancer Cells to Metabolic Perturbations in Bone Marrow Spheroids.

Malignant cells from breast cancer and other common cancers such as prostate and melanoma may persist in bone marrow as quiescent, non-dividing cells that remain viable for years or even decades before resuming proliferation to cause recurrent disease. This phenomenon, referred to clinically as tumor dormancy, poses tremendous challenges to curing patients with breast cancer. Quiescent tumor cells resist chemotherapy drugs that predominantly target proliferating cells, limiting success of neo-adjuvant and adjuvant therapies. We recently developed a 3D spheroid model of quiescent breast cancer cells in bone marrow for mechanistic and drug testing studies. We combined this model with optical imaging methods for label-free detection of cells preferentially utilizing glycolysis versus oxidative metabolism to investigate the metabolic state of co-culture spheroids with different bone marrow stromal and breast cancer cells. Through imaging and biochemical assays, we identified different metabolic states of bone marrow stromal cells that control metabolic status and flexibilities of co-cultured breast cancer cells. We tested metabolic stresses and targeted inhibition of specific metabolic pathways to identify approaches to preferentially eliminate quiescent breast cancer cells from bone marrow environments. These studies establish an integrated imaging approach to analyze metabolism in complex tissue environments to identify new metabolically-targeted cancer therapies.

Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions: EVIDENCE FOR PARTIAL ALLOSTERIC AGONISM IN COMPARISON WITH CXCL12 CHEMOKINE.

An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4.

Modeling selective elimination of quiescent cancer cells from bone marrow.

Patients with many types of malignancy commonly harbor quiescent disseminated tumor cells in bone marrow. These cells frequently resist chemotherapy and may persist for years before proliferating as recurrent metastases. To test for compounds that eliminate quiescent cancer cells, we established a new 384-well 3D spheroid model in which small numbers of cancer cells reversibly arrest in G1/G0 phase of the cell cycle when cultured with bone marrow stromal cells. Using dual-color bioluminescence imaging to selectively quantify viability of cancer and stromal cells in the same spheroid, we identified single compounds and combination treatments that preferentially eliminated quiescent breast cancer cells but not stromal cells. A treatment combination effective against malignant cells in spheroids also eliminated breast cancer cells from bone marrow in a mouse xenograft model. This research establishes a novel screening platform for therapies that selectively target quiescent tumor cells, facilitating identification of new drugs to prevent recurrent cancer.

Fluorescence Lifetime Imaging of Apoptosis.

Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools to analyze cell signaling and function at single cell resolution in standard two-dimensional cell cultures, but these reporters rarely have been applied to three-dimensional environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in absorption of light complicate this analysis method in three-dimensional settings. Here we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time, to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from two-dimensional cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity.