Comparison of Tissue Molecular Biomarker Testing Turnaround Times and Concordance Between Standard of Care and the Biocartis Idylla Platform in Patients With Colorectal Cancer.

OBJECTIVES: Management of colorectal cancer warrants mutational analysis of KRAS/NRAS when considering anti-epidermal growth factor receptor therapy and BRAF testing for prognostic stratification. In this multicenter study, we compared a fully integrated, cartridge-based system to standard-of-care assays used by participating laboratories. METHODS: Twenty laboratories enrolled 874 colorectal cancer cases between November 2017 and December 2018. Testing was performed on the Idylla automated system (Biocartis) using the KRAS and NRAS-BRAF cartridges (research use only) and results compared with in-house standard-of-care testing methods. RESULTS: There were sufficient data on 780 cases to measure turnaround time compared with standard assays. In-house polymerase chain reaction (PCR) had an average testing turnaround time of 5.6 days, send-out PCR of 22.5 days, in-house Sanger sequencing of 14.7 days, send-out Sanger of 17.8 days, in-house next-generation sequencing (NGS) of 12.5 days, and send-out NGS of 20.0 days. Standard testing had an average turnaround time of 11 days. Idylla average time to results was 4.9 days with a range of 0.4 to 13.5 days. CONCLUSIONS: The described cartridge-based system offers rapid and reliable testing of clinically actionable mutation in colorectal cancer specimens directly from formalin-fixed, paraffin-embedded tissue sections. Its simplicity and ease of use compared with other molecular techniques make it suitable for routine clinical laboratory testing.

BCL-2 expression aids in the immunohistochemical prediction of the Oncotype DX breast cancer recurrence score.

BACKGROUND: The development of molecular techniques to estimate the risk of breast cancer recurrence has been a significant addition to the suite of tools available to pathologists and breast oncologists. It has previously been shown that immunohistochemistry can provide a surrogate measure of tumor recurrence risk, effectively providing a less expensive and more rapid estimate of risk without the need for send-out. However, concordance between gene expression-based and immunohistochemistry-based approaches has been modest, making it difficult to determine when one approach can serve as an adequate substitute for the other. We investigated whether immunohistochemistry-based methods can be augmented to provide a useful therapeutic indicator of risk. METHODS: We studied whether the Oncotype DX breast cancer recurrence score can be predicted from routinely acquired immunohistochemistry of breast tumor histology. We examined the effects of two modifications to conventional scoring measures based on ER, PR, Ki-67, and Her2 expression. First, we tested a mathematical transformation that produces a more diagnostic-relevant representation of the staining attributes of these markers. Second, we considered the expression of BCL-2, a complex involved in regulating apoptosis, as an additional prognostic marker. RESULTS: We found that the mathematical transformation improved concordance rates over the conventional scoring model. By establishing a measure of prediction certainty, we discovered that the difference in concordance between methods was even greater among the most certain cases in the sample, demonstrating the utility of an accompanying measure of prediction certainty. Including BCL-2 expression in the scoring model increased the number of breast cancer cases in the cohort that were considered high certainty, effectively expanding the applicability of this technique to a greater proportion of patients. CONCLUSIONS: Our results demonstrate an improvement in concordance between immunohistochemistry-based and gene expression-based methods to predict breast cancer recurrence risk following two simple modifications to the conventional scoring model.

Estimation of Fine-Scale Histologic Features at Low Magnification.

CONTEXT.­: Whole-slide imaging has ushered in a new era of technology that has fostered the use of computational image analysis for diagnostic support and has begun to transfer the act of analyzing a slide to computer monitors. Due to the overwhelming amount of detail available in whole-slide images, analytic procedures-whether computational or visual-often operate at magnifications lower than the magnification at which the image was acquired. As a result, a corresponding reduction in image resolution occurs. It is unclear how much information is lost when magnification is reduced, and whether the rich color attributes of histologic slides can aid in reconstructing some of that information. OBJECTIVE.­: To examine the correspondence between the color and spatial properties of whole-slide images to elucidate the impact of resolution reduction on the histologic attributes of the slide. DESIGN.­: We simulated image resolution reduction and modeled its effect on classification of the underlying histologic structure. By harnessing measured histologic features and the intrinsic spatial relationships between histologic structures, we developed a predictive model to estimate the histologic composition of tissue in a manner that exceeds the resolution of the image. RESULTS.­: Reduction in resolution resulted in a significant loss of the ability to accurately characterize histologic components at magnifications less than ×10. By utilizing pixel color, this ability was improved at all magnifications. CONCLUSIONS.­: Multiscale analysis of histologic images requires an adequate understanding of the limitations imposed by image resolution. Our findings suggest that some of these limitations may be overcome with computational modeling.

Invasive breast carcinomas with ATM gene variants of uncertain significance share distinct histopathologic features.

The increasing availability of next-generation sequencing for clinical research dramatically improved our understanding of breast cancer genetics and resulted in detection of new mutation variants. Cancer risk data relating to some of these variants are insufficient, prompting the designation of variants of uncertain significance (VUS). The histopathologic characteristics of these variants have not been previously described. We propose to depict these characteristics and determine if invasive carcinomas with similar VUS genes share similar histomorphologic features. In total, 28 invasive breast cancers with VUS were retrospectively identified. Tumor sections were reviewed and a predefined set of histopathologic characteristics were documented and compared. Nine of the 28 cases were variants in the ATM gene and were found to share similar histologic characteristics; all had tumor cells with low nuclear grade, absent tumor infiltrating lymphocytes, as well as a marked desmoplastic response. A subset of the above findings were identified in variants of other genes but none had all findings collectively. Furthermore, variants of ATM gene had smaller tumor size, lower pathologic T stage at presentation, and more favorable surrogate molecular subtype compared to variants of other genes. These findings could potentially be used to reclassify VUS and predict which patients may harbor ATM mutations, and hence could have implications in triaging toward ATM variant identification for potential future targeted therapy.

An alternative reference space for H&E color normalization.

Digital imaging of H&E stained slides has enabled the application of image processing to support pathology workflows. Potential applications include computer-aided diagnostics, advanced quantification tools, and innovative visualization platforms. However, the intrinsic variability of biological tissue and the vast differences in tissue preparation protocols often lead to significant image variability that can hamper the effectiveness of these computational tools. We developed an alternative representation for H&E images that operates within a space that is more amenable to many of these image processing tools. The algorithm to derive this representation operates by exploiting the correlation between color and the spatial properties of the biological structures present in most H&E images. In this way, images are transformed into a structure-centric space in which images are segregated into tissue structure channels. We demonstrate that this framework can be extended to achieve color normalization, effectively reducing inter-slide variability.

Novel Small-Molecule CX3CR1 Antagonist Impairs Metastatic Seeding and Colonization of Breast Cancer Cells.

UNLABELLED: Recent evidence indicates that cancer cells, even in the absence of a primary tumor, recirculate from established secondary lesions to further seed and colonize skeleton and soft tissues, thus expanding metastatic dissemination and precipitating the clinical progression to terminal disease. Recently, we reported that breast cancer cells utilize the chemokine receptor CX3CR1 to exit the blood circulation and lodge to the skeleton of experimental animals. Now, we show that CX3CR1 is overexpressed in human breast tumors and skeletal metastases. To assess the clinical potential of targeting CX3CR1 in breast cancer, a functional role of CX3CR1 in metastatic seeding and progression was first validated using a neutralizing antibody for this receptor and transcriptional suppression by CRISPR interference (CRISPRi). Successively, we synthesized and characterized JMS-17-2, a potent and selective small-molecule antagonist of CX3CR1, which was used in preclinical animal models of seeding and established metastasis. Importantly, counteracting CX3CR1 activation impairs the lodging of circulating tumor cells to the skeleton and soft-tissue organs and also negatively affects further growth of established metastases. Furthermore, nine genes were identified that were similarly altered by JMS-17-2 and CRISPRi and could sustain CX3CR1 prometastatic activity. In conclusion, these data support the drug development of CX3CR1 antagonists, and promoting their clinical use will provide novel and effective tools to prevent or contain the progression of metastatic disease in breast cancer patients. IMPLICATIONS: This work conclusively validates the instrumental role of CX3CR1 in the seeding of circulating cancer cells and is expected to pave the way for pairing novel inhibitors of this receptor with current standards of care for the treatment of breast cancer patients. Mol Cancer Res; 14(6); 518-27. ©2016 AACR.

An optimized color transformation for the analysis of digital images of hematoxylin & eosin stained slides.

Hematoxylin and eosin (H&E) staining is ubiquitous in pathology practice and research. As digital pathology has evolved, the reliance of quantitative methods that make use of H&E images has similarly expanded. For example, cell counting and nuclear morphometry rely on the accurate demarcation of nuclei from other structures and each other. One of the major obstacles to quantitative analysis of H&E images is the high degree of variability observed between different samples and different laboratories. In an effort to characterize this variability, as well as to provide a substrate that can potentially mitigate this factor in quantitative image analysis, we developed a technique to project H&E images into an optimized space more appropriate for many image analysis procedures. We used a decision tree-based support vector machine learning algorithm to classify 44 H&E stained whole slide images of resected breast tumors according to the histological structures that are present. This procedure takes an H&E image as an input and produces a classification map of the image that predicts the likelihood of a pixel belonging to any one of a set of user-defined structures (e.g., cytoplasm, stroma). By reducing these maps into their constituent pixels in color space, an optimal reference vector is obtained for each structure, which identifies the color attributes that maximally distinguish one structure from other elements in the image. We show that tissue structures can be identified using this semi-automated technique. By comparing structure centroids across different images, we obtained a quantitative depiction of H&E variability for each structure. This measurement can potentially be utilized in the laboratory to help calibrate daily staining or identify troublesome slides. Moreover, by aligning reference vectors derived from this technique, images can be transformed in a way that standardizes their color properties and makes them more amenable to image processing.

Lymph Node Metastasis Status in Breast Carcinoma Can Be Predicted via Image Analysis of Tumor Histology.

OBJECTIVE: To develop a method whereby axillary lymph node (ALN) metastasis can be predicted without ALN dissection, via computational image analysis of routinely acquired tumor histology. STUDY DESIGN: We employed digital image processing to stratify patients based on the histological attributes of the primary tumor. We extracted image features that capture the nuclear and architectural properties of the specimen. We then used a novel machine learning algorithm to transform image features into a scalar score that provided not only a metastasis prediction but also the certainty of classification. RESULTS: We applied this procedure to 101 patients with a ground truth established by histological examination of the lymph nodes and found that 68.3% of the cohort could be classified, exhibiting a correct prediction rate of 88.4%. CONCLUSION: These results demonstrate a technique that potentially can be used to supplant existing surgical methods to determine ALN metastasis status, thereby reducing patient morbidity associated with over-treatment.

Interleukin-1ß promotes skeletal colonization and progression of metastatic prostate cancer cells with neuroendocrine features.

Despite the progress made in the early detection and treatment of prostate adenocarcinoma, the metastatic lesions from this tumor are incurable. We used genome-wide expression analysis of human prostate cancer cells with different metastatic behavior in animal models to reveal that bone-tropic phenotypes upregulate three genes encoding for the cytokine interleukin-1ß (IL-1ß), the chemokine CXCL6 (GCP-2), and the protease inhibitor elafin (PI3). The Oncomine database revealed that these three genes are significantly upregulated in human prostate cancer versus normal tissue and correlate with Gleason scores ≥7. This correlation was further validated for IL-1ß by immunodetection in prostate tissue arrays. Our study also shows that the exogenous overexpression of IL-1ß in nonmetastatic cancer cells promotes their growth into large skeletal lesions in mice, whereas its knockdown significantly impairs the bone progression of highly metastatic cells. In addition, IL-1ß secreted by metastatic cells induced the overexpression of COX-2 (PTGS2) in human bone mesenchymal cells treated with conditioned media from bone metastatic prostate cancer cells. Finally, we inspected human tissue specimens from skeletal metastases and detected prostate cancer cells positive for both IL-1ß and synaptophysin while concurrently lacking prostate-specific antigen (PSA, KLK3) expression. Collectively, these findings indicate that IL-1ß supports the skeletal colonization and metastatic progression of prostate cancer cells with an acquired neuroendocrine phenotype.

Astrocyte senescence as a component of Alzheimer's disease.

Aging is the main risk factor for Alzheimer's disease (AD); however, the aspects of the aging process that predispose the brain to the development of AD are largely unknown. Astrocytes perform a myriad of functions in the central nervous system to maintain homeostasis and support neuronal function. In vitro, human astrocytes are highly sensitive to oxidative stress and trigger a senescence program when faced with multiple types of stress. In order to determine whether senescent astrocytes appear in vivo, brain tissue from aged individuals and patients with AD was examined for the presence of senescent astrocytes using p16(INK4a) and matrix metalloproteinase-1 (MMP-1) expression as markers of senescence. Compared with fetal tissue samples (n = 4), a significant increase in p16(INK4a)-positive astrocytes was observed in subjects aged 35 to 50 years (n = 6; P = 0.02) and 78 to 90 years (n = 11; P<10(-6)). In addition, the frontal cortex of AD patients (n = 15) harbored a significantly greater burden of p16(INK4a)-positive astrocytes compared with non-AD adult control subjects of similar ages (n = 25; P = 0.02) and fetal controls (n = 4; P<10(-7)). Consistent with the senescent nature of the p16(INK4a)-positive astrocytes, increased metalloproteinase MMP-1 correlated with p16(INK4a). In vitro, beta-amyloid 1-42 (Aß(1-42)) triggered senescence, driving the expression of p16(INK4a) and senescence-associated beta-galactosidase. In addition, we found that senescent astrocytes produce a number of inflammatory cytokines including interleukin-6 (IL-6), which seems to be regulated by p38MAPK. We propose that an accumulation of p16(INK4a)-positive senescent astrocytes may link increased age and increased risk for sporadic AD.

Left atrial fibroelastoma.

We describe a 67-year-old man with a history of stroke who was found to have a mass at the left atrial ridge, at the free wall of the left atrium between the left atrial appendage and the pulmonary vein. The mass was removed surgically and pathological analysis showed fibroelastoma. A literature search showed that fibroelastoma in the left atrial ridge frequently causes embolic stroke.

A visual analytics system for breast tumor evaluation.

OBJECTIVE: To develop a system for the interactive exploration and examination of histologically derived data that is associated with breast tumors and may be used to evaluate the histologic grade of the tumor. STUDY DESIGN: The system integrates pathologist-generated prognostic data with 2-dimensional (2-D) image analysis data, 2-D digital tissue cross-sections and annotations, 3-dimensional (3-D) tumor reconstructions and volumetric analysis, 3D spatial tumor display and recorded prognostic information from available cases in the Drexel University College of Medicine tumor databank. The system consists of 3 components: (1) a user interface for applying 2-D image processing, segmentation and annotation to a digitized histology slide, (2) a distance field interpolation method for contour-based 3D reconstruction of breast tumors and volumetric model analysis routines and (3) a Web-based database management interface for interactive data browsing and searching and multimodality visualization. RESULTS: The system has been implemented and deployed with data from 36 breast cancer cases, 7 of which have been reconstructed in 3-D. CONCLUSION: Interactive visual analytics technology may be used to create an effective breast tumor evaluation system.

A study of shape distributions for estimating histologic grade.

Breast cancers can be histologically categorized (graded) based upon their architectural patterns and cellular types. Inaccurate histologic grading can result in inappropriate treatment for a given patient. Computational analysis of breast cancers offers an operator-independent method for histologic grading that should enhance grading reliability. We present the initial efforts to develop computational technologies that may be used to automatically and objectively estimate the histologic grade of breast cancer tumors. The approach utilizes image processing and shape analysis of imaged histologic sections. Our work is based on the hypothesis that cellular structures found in breast cancer tumors can be transformed into distinct high-resolution shape distributions using geometric measures from stochastic geometry. The resulting shape distributions define well-populated regions of the associated high-dimensional space. Mapping an unknown breast cancer sample into this high-D space and determining to which region it belongs will allow for the automatic estimation of its histologic grade.

Increased sensitivity of anal cytology in evaluation of internal compared with external lesions.

OBJECTIVE: To investigate the impact of keratin on the accuracy of internal and external anal brush sampling of known lesions. STUDY DESIGN: A group of 46 human immunodeficiency virus (HIV)-seropositive patients underwent external and internal anal brush sampling before biopsy of known lesions. RESULTS; A total of 92 ThinPrep (46 external, 46 internal) an 211 biopsies were examined. The sensitivity and specificity for internal lesions positive and negative for anal squa mous intraepithelial lesion (ASIL) was 91.1% and 42.8%, respectively; and for external lesions was 79.4% and 100%, respectively. Low cellularity on cytology and markedly thickened keratin on biopsy were significantly more common in external compared with internal lesions (p < 0.0001). CONCLUSION: We conclude that hyperkeratosis interferes with adequate sampling and accurate grading of external anal lesions by brush sampling.

Prostatic basal cells in the peripheral and transitional zones: zonal variation in morphology and in immunophenotype.

BACKGROUND: Benign prostatic hyperplasia and prostatic adenocarcinoma exhibit prominent zonal predilections. Basal cells from the transitional zone and from the peripheral zone are postulated to have different underlying biological properties. We studied basal cells in both prostatic zones. METHODS: Tissue microarrays (TMA) were prepared from 65 whole-mounted prostatectomy specimens with prostatic adenocarcinoma. The transitional zone and peripheral zone were sampled from each prostate. TMA sections were stained with a basal cell cocktail (CK 34betaE12 + p63). The immunostaining pattern and the morphology of basal cells were recorded. RESULTS: Triangular-shaped basal cells were highlighted by CK 34betaE12 cytoplasmic and p63 nuclear staining. These basal cells had their long axis oriented perpendicular to the basement membrane and their apex toward the lumen interdigited between secretory luminal cells. This morphology was seen in the majority of peripheral zone benign prostatic glands (92.0%) but only a minority of transitional zone benign prostatic glands (18.0%). Basal cells of the transitional zone showed weak or absent CK 34betaE12 staining in 65.9% of glands while maintaining p63. All glands with high-grade prostatic intraepithelial neoplasia (HGPIN) contained the triangular basal cells. In addition, basal cell clusters were identified in 8.7% of peripheral zone glands and 5.2% of HGPIN glands. CONCLUSIONS: Our results indicate that the basal cell morphology and the basal cell immunophenotype have a zonal variation. The finding of a unique morphology of basal cells and the presence of basal cell clusters in the peripheral zone suggests that the peripheral zone might be the stem/progenitor cell-rich area in the human prostates.

Method for quantification of a prostate cancer biomarker in urine without sample preparation.

We describe a macrocantilever-based method for detecting a prostate cancer biomarker (alpha-methylacyl-CoA racemase; AMACR) directly in patient urine without a sample preparation step and without the use of labeled reagents. Clean catch voided urine specimens were prospectively collected from five confirmed prostate cancer patients 3 weeks postbiopsy. The presence of AMACR was measured in a blinded manner by exposing 3 mL of urine to the anti-AMACR-immobilized piezoelectric-excited millimeter-sized (PEMC) sensor. The resonance frequency of PEMC decreases as AMACR from sample binds to the antibody on the sensor. The resonance frequency changes for the five patients tested were 4,314 +/- 35 (n = 2), 269 +/- 17 (n = 2), 977 +/- 64 (n = 3), 600 +/- 31 (n = 2), and 801 +/- 81 (n = 2) Hz, respectively. Positive detection was observed within approximately 15 min. The responses to positive, negative, and buffer controls were -9 +/- 13, -34 +/- 18, and -6 +/- 18 Hz, respectively. Positive verification of AMACR attachment was confirmed by low-pH buffer release. The sensor response was quantitatively related to AMACR concentration in control urine, and the relationship was used in developing an in situ calibration method for quantifying AMACR in patient urine. Estimated concentrations of 42, 2, and 3 fg/mL AMACR were calculated for the three patients' urine, while absence of AMACR was confirmed in control urine (n = 13). Because of simplicity of measurement combined with high sensitivity and specificity, the method may be a useful adjunct in a point-of-care setting to identify men at increased risk for prostate cancer.

Insulin-like growth factor-I produced by seminal vesicles: relationship to intraepithelial basal cell hyperplasia in the prostate.

PURPOSE: This study examined the seminal vesicle fluid (SVF) as a potential local source of insulin-like growth factor-I (IGF-I) in the peripheral zone of the prostate. EXPERIMENTAL DESIGN: IGF-I levels in seminal fluid were measured. The levels of the IGF-I receptor (IGF-IR) in its active, phosphorylated form as well as direct downstream targets were examined in the peripheral zone of the prostate. RESULTS: In situ, we find that the IGF-IR is activated in the peripheral zone in areas of atrophy, prostatic intraepithelial hyperplasia, and cancer. In addition, immunostaining reveals preferential activation of the IGF-IR in p63-positive cells in areas of intermediate basal cell hyperplasia in the peripheral zone, indicating that prostate progenitor cells are highly sensitive to increases in local IGF-I levels. These areas of basal cell hyperplasia occur at high incidence in the peripheral zone of the prostate. Relatively high levels of IGF-I were identified in SVF. In addition, we find that SVF can stimulate the proliferation of both normal and cancer-derived prostate cells. CONCLUSIONS: These results suggest that SVF is a local source of IGF-I that provides chronic stimulation of prostate cells. This chronic stimulation could contribute to the development of prostate cancer in older men.

The ABCA5 protein: a urine diagnostic marker for prostatic intraepithelial neoplasia.

PURPOSE: To develop a urine diagnostic test for preneoplastic intraepithelial neoplasia of the prostate. EXPERIMENTAL DESIGN: We have used a DNA-binding assay and electrophoretic mobility shift assays (EMSA) to screen for novel duplexed DNA-binding sequences, which bind protein(s) overexpressed in crude protein extracts from high-grade prostatic intraepithelial neoplasia (HGPIN). EMSAs, immunohistochemistry, and ELISAs were used to measure expression of the ABCA5 protein identified as a specific marker in prostate tissue and patient urine. RESULTS: Following screening of 4,096 sequences, an 8-bp dsDNA sequence (i.e., TCCAGCGA) was identified, which binds the ABCA5 protein, a member of the ATP-binding cassette multidrug resistant family. EMSAs showed that ABCA5 was overexpressed in HGPIN tissue (n=11/11) and in the urine of patients with HGPIN (n=18/18) but was not expressed in prostate cancer, benign prostatic hyperplasia, or stroma. Immunohistochemistry indicated that ABCA5 was overexpressed in foci of intermediate basal cells in normal glands and in HGPIN. ABCA5 was faintly expressed in prostate cancer glands. ELISAs showed in 'blinded studies' that ABCA5 was a highly sensitive (>98% sensitivity) urine diagnostic marker for HGPIN in biopsy-positive patients (n=107) at a 'cutoff' of 25 ng/mL. ABCA5 was present at very low levels (i.e., <25 ng/mL) in the urine of patients diagnosed with benign prostatic hyperplasia (n=79) or prostatitis or kidney and bladder cancer (>86% specificity). CONCLUSIONS: The data indicate that ABCA5 might be a specific urine marker for diagnosis of patients with HGPIN.

Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery.

Breast tumors are typically heterogeneous and contain diverse subpopulations of tumor cells with differing phenotypic properties. Planar cultures of cancer cell lines are not viable models of investigation of cell-cell and cell-matrix interactions during tumor development. This article presents an in vitro coculture-based 3-dimensional heterogeneous breast tumor model that can be used in drug resistance and drug delivery investigations. Breast cancer cell lines of different phenotypes (MDAMB231, MCF7, and ZR751) were cocultured in a rotating wall vessel bioreactor to form a large number of heterogeneous tumoroids in a single cell culture experiment. Cells in the rotating vessels were labeled with Cell Tracker fluorescent probes to allow for time course fluorescence microscopy to monitor cell aggregation. Histological sections of tumoroids were stained with hematoxylin and eosin, progesterone receptor, E-cadherin (E-cad), and proliferation marker ki67. In vitro tumoroids developed in this study recapture important features of the temporal-spatial organization of solid tumors, including the presence of necrotic areas at the center and higher levels of cell division at the tumor periphery. E-cad-positive MCF7 cells form larger tumoroids than E-cad-negative MDAMB231 cells. In heterogeneous tumors, the irregular surface roughness was mainly due to the presence of MDAMB231 cells, whereas MCF7 cells formed smooth surfaces. Moreover, when heterogeneous tumoroids were placed onto collagen gels, highly invasive MDAMB231 cell-rich surface regions produced extensions into the matrix, whereas poorly invasive MCF7 cells did not. The fact that one can form a large number of 1-mm tumoroids in 1 coculture attests to the potential use of this system at high-throughput investigations of cancer drug development and drug delivery into the tumor.