Results of a phase I/IIa trial of SV-BR-1-GM inoculation with low-dose cyclophosphamide and interferon alpha (Bria-IMT) in metastatic breast cancer.

This Phase I/IIa open-label, single-arm clinical trial addressing advanced, refractory, metastatic breast cancer was conducted at six medical centers in the United States. We repeated inoculations with irradiated SV-BR-1-GM, a breast cancer cell line with antigen-presenting activity engineered to release granulocyte-macrophage colony-stimulating factor (GM-CSF), with pre-dose low-dose cyclophosphamide and post-dose local interferon alpha. Twenty-six patients were enrolled; 23 (88.5%) were inoculated, receiving a total of 79 inoculations. There were six Grade 4 and one Grade 5 adverse events noted (judged unrelated to SV-BR-1-GM). Disease control (stable disease [SD]) occurred in 8 of 16 evaluable patients; 4 showed objective regression of metastases, including 1 patient with near-complete regressions in 20 of 20 pulmonary lesions. All patients with regressions had human leukocyte antigen (HLA) matches with SV-BR-1-GM; non-responders were equally divided between matching and nonmatching (p = .01, Chi-squared), and having ≥2 HLA matches with SV-BR-1-GM (n = 6) correlated with clinical benefit. Delayed-type hypersensitivity (DTH) testing to candida antigen and SV-BR-1-GM generated positive responses (≥5 mm) in 11 (42.3%) and 13 (50%) patients, respectively. Quantifying peripheral circulating tumor cells (CTCs) and cancer-associated macrophage-like cells (CAMLs) showed that a drop in CAMLs was significantly correlated with an improvement in progression-free survival (PFS; 4.1 months vs. 1.8 months, p = .0058). Eight of 10 patients significantly upregulated programmed cell death ligand 1 (PD-L1) on CTCs/CAMLs with treatment (p = .0012). These observations support the safety of the Bria-IMT regimen, demonstrate clinical regressions, imply a role for HLA matching, and identify a possible value for monitoring CAMLs in peripheral blood.

In situtumor model for longitudinal in silico imaging trials.

Objective.In this article, we introduce a computational model for simulating the growth of breast cancer lesions accounting for the stiffness of surrounding anatomical structures.Approach.In our model, ligaments are classified as the most rigid structures while the softer parts of the breast are occupied by fat and glandular tissues As a result of these variations in tissue elasticity, the rapidly proliferating tumor cells are met with differential resistance. It is found that these cells are likely to circumvent stiffer terrains such as ligaments, instead electing to proliferate preferentially within the more yielding confines of the breast's soft topography. By manipulating the interstitial tumor pressure in direct proportion to the elastic constants of the tissues surrounding the tumor, this model thus creates the potential for realizing a database of unique lesion morphology sculpted by the distinctive topography of each local anatomical infrastructure. We modeled the growth of simulated lesions within volumes extracted from fatty breast models, developed by Graffet alwith a resolution of 50µm generated with the open-source and readily available Virtual Imaging Clinical Trials for Regulatory Evaluation (VICTRE) imaging pipeline. To visualize and validate the realism of the lesion models, we leveraged the imaging component of the VICTRE pipeline, which replicates the siemens mammomat inspiration mammography system in a digital format. This system was instrumental in generating digital mammogram (DM) images for each breast model containing the simulated lesions.Results.By utilizing the DM images, we were able to effectively illustrate the imaging characteristics of the lesions as they integrated with the anatomical backgrounds. Our research also involved a reader study that compared 25 simulated DM regions of interest (ROIs) with inserted lesions from our models with DM ROIs from the DDSM dataset containing real manifestations of breast cancer. In general the simulation time for the lesions was approximately 2.5 hours, but it varied depending on the lesion's local environment.Significance.The lesion growth model will facilitate and enhance longitudinal in silico trials investigating the progression of breast cancer.

Assessing satisfaction of search in virtual mammograms for experienced and novice searchers.

Purpose: Satisfaction of search (SOS) is a phenomenon where searchers are more likely to miss a lesion/target after detecting a first lesion/target. Here, we investigated SOS for masses and calcifications in virtual mammograms with experienced and novice searchers to determine the extent to which: (1) SOS affects breast lesion detection, (2) similarity between lesions impacts detection, and (3) experience impacts SOS rates. Approach: The open virtual clinical trials framework was used to simulate the breast anatomy of patients, and up to two simulated masses and/or single-calcifications were inserted into the breast models. Experienced searchers (residents, fellows, and radiologists with breast imaging experience) and novice searchers (undergraduates who had no breast imaging experience) were instructed to search for up to two lesions (masses and calcifications) per image. Results: 2×2 mixed factors analysis of variances (ANOVAs) were run with: (1) single versus second lesion hit rates, (2) similar versus dissimilar second-lesion hit rates, and (3) similar versus dissimilar second-lesion response times as within-subject factors and experience as the between subject's factor. The ANOVAs demonstrated that: (1) experienced and novice searchers made a significant amount of SOS errors, (2) similarity had little impact on experienced searchers, but novice searchers were more likely to miss a dissimilar second lesion compared to when it was similar to a detected first lesion, (3) experienced and novice searchers were faster at finding similar compared to dissimilar second lesions. Conclusions: We demonstrated that SOS is a significant cause of lesion misses in virtual mammograms and that reader experience impacts detection rates for similar compared to dissimilar abnormalities. These results suggest that experience may impact strategy and/or recognition with theoretical implications for determining why SOS occurs.

A 2D Synthesized Image Improves the 3D Search for Foveated Visual Systems.

Current medical imaging increasingly relies on 3D volumetric data making it difficult for radiologists to thoroughly search all regions of the volume. In some applications (e.g., Digital Breast Tomosynthesis), the volumetric data is typically paired with a synthesized 2D image (2D-S) generated from the corresponding 3D volume. We investigate how this image pairing affects the search for spatially large and small signals. Observers searched for these signals in 3D volumes, 2D-S images, and while viewing both. We hypothesize that lower spatial acuity in the observers' visual periphery hinders the search for the small signals in the 3D images. However, the inclusion of the 2D-S guides eye movements to suspicious locations, improving the observer's ability to find the signals in 3D. Behavioral results show that the 2D-S, used as an adjunct to the volumetric data, improves the localization and detection of the small (but not large) signal compared to 3D alone. There is a concomitant reduction in search errors as well. To understand this process at a computational level, we implement a Foveated Search Model (FSM) that executes human eye movements and then processes points in the image with varying spatial detail based on their eccentricity from fixations. The FSM predicts human performance for both signals and captures the reduction in search errors when the 2D-S supplements the 3D search. Our experimental and modeling results delineate the utility of 2D-S in 3D search-reduce the detrimental impact of low-resolution peripheral processing by guiding attention to regions of interest, effectively reducing errors.

Computational Breast Anatomy Simulation Using Multi-Scale Perlin Noise.

Virtual clinical trials (VCTs) of medical imaging require realistic models of human anatomy. For VCTs in breast imaging, a multi-scale Perlin noise method is proposed to simulate anatomical structures of breast tissue in the context of an ongoing breast phantom development effort. Four Perlin noise distributions were used to replace voxels representing the tissue compartments and Cooper's ligaments in the breast phantoms. Digital mammography and tomosynthesis projections were simulated using a clinical DBT system configuration. Power-spectrum analyses and higher-order statistics properties using Laplacian fractional entropy (LFE) of the parenchymal texture are presented. These objective measures were calculated in phantom and patient images using a sample of 140 clinical mammograms and 500 phantom images. Power-law exponents were calculated using the slope of the curve fitted in the low frequency [0.1, 1.0] mm-1 region of the power spectrum. The results show that the images simulated with our prior and proposed Perlin method have similar power-law spectra when compared with clinical mammograms. The power-law exponents calculated are -3.10, -3.55, and -3.46, for the log-power spectra of patient, prior phantom and proposed phantom images, respectively. The results also indicate an improved agreement between the mean LFE estimates of Perlin-noise based phantoms and patients than our prior phantoms and patients. Thus, the proposed method improved the simulation of anatomic noise substantially compared to our prior method, showing close agreement with breast parenchyma measures.

Under-exploration of Three-Dimensional Images Leads to Search Errors for Small Salient Targets.

Advances in 3D imaging technology are transforming how radiologists search for cancer1,2 and how security officers scrutinize baggage for dangerous objects.3 These new 3D technologies often improve search over 2D images4,5 but vastly increase the image data. Here, we investigate 3D search for targets of various sizes in filtered noise and digital breast phantoms. For a Bayesian ideal observer optimally processing the filtered noise and a convolutional neural network processing the digital breast phantoms, search with 3D image stacks increases target information and improves accuracy over search with 2D images. In contrast, 3D search by humans leads to high miss rates for small targets easily detected in 2D search, but not for larger targets more visible in the visual periphery. Analyses of human eye movements, perceptual judgments, and a computational model with a foveated visual system suggest that human errors can be explained by interaction among a target's peripheral visibility, eye movement under-exploration of the 3D images, and a perceived overestimation of the explored area. Instructing observers to extend the search reduces 75% of the small target misses without increasing false positives. Results with twelve radiologists confirm that even medical professionals reading realistic breast phantoms have high miss rates for small targets in 3D search. Thus, under-exploration represents a fundamental limitation to the efficacy with which humans search in 3D image stacks and miss targets with these prevalent image technologies.

Measurement of the useful field of view for single slices of different imaging modalities and targets.

Purpose: With three-dimensional (3-D) images displayed as stacks of 2-D images, radiologists rely more heavily on vision away from their fixation point to visually process information, guide eye movements, and detect abnormalities. Thus the ability to detect targets away from the fixation point, commonly characterized as the useful field of view (UFOV), becomes critical for these 3-D imaging modalities. We investigate how the UFOV, defined as the eccentricity, in which detection performance degrades to a given probability, varies across imaging modalities and targets. Approach: We measure the detectability of different targets at various distances from gaze locations for single slices of liver computed tomography (CT), 2-D digital mammograms (DM), and single slices of digital breast tomosynthesis (DBT) cases. Observers with varying expertise were instructed to maintain their gaze at a point while a short display of the image was flashed and an eye tracker verified observer's steady fixation. Display times were 200 and 1000 ms for CT images and 500 ms for DM and DBT images. Results: We find variations in the UFOV from 9 to 12 deg for liver CT to as small as 2.5 to 5 deg for calcification clusters in breast images (DM and DBT). We compare our results to those reported in the literature for lung nodules and discuss the differences across methods used to measure the UFOV, their dependence on case selection/task difficulty, viewing conditions, and observer expertise. We propose a complementary measure defined in terms of performance degradation relative to the peak foveal performance (relative-UFOV) to circumvent UFOV's variations with case selection/task difficulty. Conclusion: Our results highlight the variations in the UFOV across imaging modalities, target types, observer expertise, and measurement methods and suggest an additional relative-UFOV measure to more thoroughly characterize the detection performance away from point of fixation.

Author Correction: The Rho GTPase Rnd1 suppresses mammary tumorigenesis and EMT by restraining Ras-MAPK signalling.

In the version of this Article originally published the same blot was inadvertently presented as both p-Rb and Cyclin A in Fig. 2a. This blot corresponds to the p-Rb panel, as can be seen in the unprocessed version of these blots in Supplementary Fig. 9. The corrected version of the panel is shown below, together with a completely uncropped image of both blots. In addition, in the 'Viral transduction' section of the Methods, the pLKO.1 plasmids encoding short hairpin RNAs targeting human Rnd1 were incorrectly listed as clones TRCN0000018338 and TRCN0000039977. The correct clone numbers are TRCN0000047434 and TRCN0000047435.

Integrin-α10 Dependency Identifies RAC and RICTOR as Therapeutic Targets in High-Grade Myxofibrosarcoma.

Myxofibrosarcoma is a common mesenchymal malignancy with complex genomics and heterogeneous clinical outcomes. Through gene-expression profiling of 64 primary high-grade myxofibrosarcomas, we defined an expression signature associated with clinical outcome. The gene most significantly associated with disease-specific death and distant metastasis was ITGA10 (integrin-α10). Functional studies revealed that myxofibrosarcoma cells strongly depended on integrin-α10, whereas normal mesenchymal cells did not. Integrin-α10 transmitted its tumor-specific signal via TRIO and RICTOR, two oncoproteins that are frequently co-overexpressed through gene amplification on chromosome 5p. TRIO and RICTOR activated RAC/PAK and AKT/mTOR to promote sarcoma cell survival. Inhibition of these proteins with EHop-016 (RAC inhibitor) and INK128 (mTOR inhibitor) had antitumor effects in tumor-derived cell lines and mouse xenografts, and combining the drugs enhanced the effects. Our results demonstrate the importance of integrin-α10/TRIO/RICTOR signaling for driving myxofibrosarcoma progression and provide the basis for promising targeted treatment strategies for patients with high-risk disease. SIGNIFICANCE: Identifying the molecular pathogenesis for myxofibrosarcoma progression has proven challenging given the highly complex genomic alterations in this tumor type. We found that integrin-α10 promotes tumor cell survival through activation of TRIO-RAC-RICTOR-mTOR signaling, and that inhibitors of RAC and mTOR have antitumor effects in vivo, thus identifying a potential treatment strategy for patients with high-risk myxofibrosarcoma. Cancer Discov; 6(10); 1148-65. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.

The Rho GTPase Rnd1 suppresses mammary tumorigenesis and EMT by restraining Ras-MAPK signalling.

We identified the Rho GTPase Rnd1 as a candidate metastasis suppressor in basal-like and triple-negative breast cancer through bioinformatics analysis. Depletion of Rnd1 disrupted epithelial adhesion and polarity, induced epithelial-to-mesenchymal transition, and cooperated with deregulated expression of c-Myc or loss of p53 to cause neoplastic conversion. Mechanistic studies revealed that Rnd1 suppresses Ras signalling by activating the GAP domain of Plexin B1, which inhibits Rap1. Rap1 inhibition in turn led to derepression of p120 Ras-GAP, which was able to inhibit Ras. Inactivation of Rnd1 in mammary epithelial cells induced highly undifferentiated and invasive tumours in mice. Conversely, Rnd1 expression inhibited spontaneous and experimental lung colonization in mouse models of metastasis. Genomic studies indicated that gene deletion in combination with epigenetic silencing or, more rarely, point mutation inactivates RND1 in human breast cancer. These results reveal a previously unappreciated mechanism through which Rnd1 restrains activation of Ras-MAPK signalling and breast tumour initiation and progression.

Genomic deregulation during metastasis of renal cell carcinoma implements a myofibroblast-like program of gene expression.

Clear cell renal cell carcinoma (RCC) is the most common and invasive adult kidney cancer. The genetic and biological mechanisms that drive metastatic spread of RCC remain largely unknown. We have investigated the molecular signatures and underlying genomic aberrations associated with RCC metastasis, using an approach that combines a human xenograft model; expression profiling of RNA, DNA, and microRNA (miRNA); functional verification; and clinical validation. We show that increased metastatic activity is associated with acquisition of a myofibroblast-like signature in both tumor cell lines and in metastatic tumor biopsies. Our results also show that the mesenchymal trait did not provide an invasive advantage to the metastatic tumor cells. We further show that some of the constituents of the mesenchymal signature, including the expression of the well-characterized myofibroblastic marker S100A4, are functionally relevant. Epigenetic silencing and miRNA-induced expression changes accounted for the change in expression of a significant number of genes, including S100A4, in the myofibroblastic signature; however, DNA copy number variation did not affect the same set of genes. These findings provide evidence that widespread genetic and epigenetic alterations can lead directly to global deregulation of gene expression and contribute to the development or progression of RCC metastasis culminating in a highly malignant myofibroblast-like cell.

Loss of the tumor suppressor gene NF2, encoding merlin, constitutively activates integrin-dependent mTORC1 signaling.

Integrin signaling promotes, through p21-activated kinase, phosphorylation and inactivation of the tumor suppressor merlin, thus removing a block to mitogenesis in normal cells. However, the biochemical function of merlin and the effector pathways critical for the pathogenesis of malignant mesothelioma and other NF2-related malignancies are not known. We report that integrin-specific signaling promotes activation of mTORC1 and cap-dependent mRNA translation. Depletion of merlin rescues mTORC1 signaling in cells deprived of anchorage to a permissive extracellular matrix, suggesting that integrin signaling controls mTORC1 through inactivation of merlin. This signaling pathway controls translation of the cyclin D1 mRNA and, thereby, cell cycle progression. In addition, it promotes cell survival. Analysis of a panel of malignant mesothelioma cell lines reveals a strong correlation between loss of merlin and activation of mTORC1. Merlin-negative lines are sensitive to the growth-inhibitory effect of rapamycin, and the expression of recombinant merlin renders them partially resistant to rapamycin. Conversely, depletion of merlin restores rapamycin sensitivity in merlin-positive lines. These results indicate that integrin-mediated adhesion promotes mTORC1 signaling through the inactivation of merlin. Furthermore, they reveal that merlin-negative mesotheliomas display unregulated mTORC1 signaling and are sensitive to rapamycin, thus providing a preclinical rationale for prospective, biomarker-driven clinical studies of mTORC1 inhibitors in these tumors.

Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane.

Introduction of activated p21-activated kinase (PAK) is sufficient to release primary endothelial cells from contact inhibition of growth. Confluent cells display deficient activation of PAK and translocation of Rac to the plasma membrane at matrix adhesions. Targeting Rac to the plasma membrane rescues these cells from contact inhibition. PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin. Merlin mutants, which are presumed to exert a dominant-negative effect, enable recruitment of Rac to matrix adhesions and promote mitogenesis in confluent cells. Small interference RNA-mediated knockdown of Merlin exerts the same effects. Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway. These results provide a framework for understanding the tumor suppressor function of Merlin and indicate that Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions.

Structural determinants for the biological activity of Vav proteins.

We have used an extensive mutagenesis approach to study the specific role of the eight structural domains of Vav during both the activation and signaling steps of this Rac1 exchange factor. Our results indicate that several Vav domains (Dbl homology, pleckstrin homology, and zinc finger) are essential for all the biological activities tested, whereas others are required for discrete, cell type-specific biological effects. Interestingly, we have found that Vav domains have no unique functions. Thus, the calponin homology domain mediates the inhibition of Vav both in vitro and in vivo but, at the same time, exerts effector functions in lymphocytes upon receptor activation. The Vav SH2 and SH3 regions play regulatory roles in the activation of Vav in fibroblasts, mediating both its phosphorylation and translocation to the plasma membrane. In contrast, the Vav SH2 and SH3 regions act as scaffolding platforms in T-cells, ensuring the proper phosphorylation of Vav and the subsequent engagement of downstream effectors. We also provide evidence indicating that the zinc finger region exerts at least three different functional roles in Vav, aiding in the down-regulation of its basal activity, the engagement of substrates, and the induction of ancillary pathways required for cell transformation. Finally, the results obtained are consistent with a new regulatory model for Vav, in which the calponin homology region inhibits the basal activity of Vav through interactions with the zinc finger region.