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Photon absorption remote sensing (PARS) is a new laser-based microscope technique that permits cellular-level resolution of unstained fresh, frozen, and fixed tissues. Our objective was to determine whether PARS could provide an image quality sufficient for the diagnostic assessment of breast cancer needle core biopsies (NCB). We PARS imaged and virtually H&E stained seven independent unstained formalin-fixed paraffin-embedded breast NCB sections. These identical tissue sections were subsequently stained with standard H&E and digitally scanned. Both the 40× PARS and H&E whole-slide images were assessed by seven breast cancer pathologists, masked to the origin of the images. A concordance analysis was performed to quantify the diagnostic performances of standard H&E and PARS virtual H&E. The PARS images were deemed to be of diagnostic quality, and pathologists were unable to distinguish the image origin, above that expected by chance. The diagnostic concordance on cancer vs. benign was high between PARS and conventional H&E (98% agreement) and there was complete agreement for within-PARS images. Similarly, agreement was substantial (kappa > 0.6) for specific cancer subtypes. PARS virtual H&E inter-rater reliability was broadly consistent with the published literature on diagnostic performance of conventional histology NCBs across all tested histologic features. PARS was able to image unstained tissues slides that were diagnostically equivalent to conventional H&E. Due to its ability to non-destructively image fixed and fresh tissues, and the suitability of the PARS output for artificial intelligence assistance in diagnosis, this technology has the potential to improve the speed and accuracy of breast cancer diagnosis. [ABSTRACT FROM AUTHOR]

Purpose: The risk assessment classification schemes for gastrointestinal stromal tumors (GIST) include tumor site, size, mitotic count and variably tumor rupture. Heterogeneity in high-risk GIST poses limitations for current classification schemes. This study aims to demonstrate the clinical utility of risk stratification by gene expression profiling (GEP) using Nanostring technology. Methods: Fifty-six GIST cases were analyzed using a 231 gene expression panel. GEP results were correlated with clinical and pathological data. The prognostic performance was assessed in 34 patients with available survival data using ROC curves, Kaplan–Meier survival curves and compared with traditional risk assessment schemes. Volcano plot analysis identified seven genes with significantly higher expression (FDR <.0.05) in high-risk than in non-high-risk tumors, namely TYMS, CDC2, TOP2A, CCNA2, E2F1, PCNA, and BIRC5. Together, these transcripts exhibited significantly higher expression in high-risk tumors than in intermediate (P < 0.01), low (P < 0.001), and very low (P = 0.01) risk tumors. Receiver-operating characteristic curve analysis demonstrated area under the curve (AUC) to be 0.858 for the separation of high-risk and non-high-risk tumors. Kaplan–Meier survival analysis demonstrated improved risk stratification (log-rank test P < 0.001) compared to the current risk assessment classification (P = 0.231). Conclusion: In addition to current clinical and histology-based risk classification for patients with GIST, gene expression may offer complementary prognostic information. [ABSTRACT FROM AUTHOR]

Purpose: N-myristoyltransferases 1 and 2 (NMT1 and NMT2) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. Myristoylation regulates numerous membrane-bound signal transduction pathways important in cancer biology and the pan-NMT inhibitor PCLX-001 is approaching clinical development as a cancer therapy. The tissue distribution, relative abundances, and prognostic value of the two human NMTs remain poorly understood. Methods: We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to human NMT1 and NMT2. These mAbs were used to perform immunohistochemical analysis of the abundance and distribution of NMT1 and NMT2 in normal breast epithelial samples and a large cohort of primary breast adenocarcinomas from the BCIRG001 clinical trial (n = 706). Results: NMT1 protein was readily quantified in normal and most transformed breast epithelial tissue and was associated with higher overall histologic grade, higher Ki67, and lower hormone receptor expression. While NMT2 protein was readily detected in normal breast epithelial tissue, it was undetectable in the majority of breast cancers. Detectable NMT2 protein correlated with significantly poorer overall survival (hazard ratio 1.36; P = 0.029) and worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. Treatment of cultured breast cancer cells with PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts produced significant dose-dependent tumor growth inhibition in vivo. Conclusions: These results support further evaluation of NMT immunohistochemistry for patient selection and clinical trials of NMT inhibition in breast cancer patients. [ABSTRACT FROM AUTHOR]

Automated cell detection and localization from microscopy images are significant tasks in biomedical research and clinical practice. In this paper, we design a new cell detection and localization algorithm that combines deep convolutional neural network (CNN) and compressed sensing (CS) or sparse coding (SC) for end-to-end training. We also derive, for the first time, a backpropagation rule, which is applicable to train any algorithm that implements a sparse code recovery layer. The key innovation behind our algorithm is that the cell detection task is structured as a point object detection task in computer vision, where the cell centers (i.e., point objects) occupy only a tiny fraction of the total number of pixels in an image. Thus, we can apply compressed sensing (or equivalently SC) to compactly represent a variable number of cells in a projected space. Subsequently, CNN regresses this compressed vector from the input microscopy image. The SC/CS recovery algorithm (${L} _{1}$ optimization) can then recover sparse cell locations from the output of CNN. We train this entire processing pipeline end-to-end and demonstrate that end-to-end training improves accuracy over a training paradigm that treats CNN and CS-recovery layers separately. We have validated our algorithm on five benchmark datasets with excellent results. [ABSTRACT FROM AUTHOR]

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We have previously identified a novel phenotypic dichotomy in breast cancer (BC) based on the response to a SRR2 (Sox2 regulatory region 2) reporter, with reporter responsive (RR) cells being more tumorigenic/stem-like than reporter unresponsive (RU) cells. Since the expression level of Sox2 is comparable between the two cell subsets, we hypothesized that post-translational modifications of Sox2 contribute to their differential reporter response and phenotypic differences. By liquid chromatography-mass spectrometry, we found Sox2 to be phosphorylated in RR but not RU cells. Threonine 116 is an important phosphorylation site, since transfection of the T116A mutant into RR cells significantly decreased the SRR2 reporter luciferase activity and the RR-associated phenotype. Oxidative stress-induced conversion of RU into RR cells was accompanied by Sox2 phosphorylation at T116 and increased Sox2-DNA binding. In a cohort of BC, we found significant correlations between the proportion of tumor cells immuno-reactive with anti-phosphorylated Sox2T116 and a high tumor grade (p = 0.006), vascular invasion (p = 0.001) and estrogen receptor expression (p = 0.032). In conclusion, our data suggests that phosphorylation of Sox2T116 contributes to the tumorigenic/stem-like features in RR cells. Detection of phospho-Sox2T116 may be useful in identifying a small subset of tumor cells carrying stem-like/tumorigenic features in BC. [ABSTRACT FROM AUTHOR]

Ki67 is a commonly used marker of cancer cell proliferation, and has significant prognostic value in breast cancer. In spite of its clinical importance, assessment of Ki67 remains a challenge, as current manual scoring methods have high inter- and intra-user variability. A major reason for this variability is selection bias, in that different observers will score different regions of the same tumor. Here, we developed an automated Ki67 scoring method that eliminates selection bias, by using whole-slide analysis to identify and score the tumor regions with the highest proliferative rates. The Ki67 indices calculated using this method were highly concordant with manual scoring by a pathologist (Pearson’s r = 0.909) and between users (Pearson’s r = 0.984). We assessed the clinical validity of this method by scoring Ki67 from 328 whole-slide sections of resected early-stage, hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. All patients had Oncotype DX testing performed (Genomic Health) and available Recurrence Scores. High Ki67 indices correlated significantly with several clinico-pathological correlates, including higher tumor grade (1 versus 3, P<0.001), higher mitotic score (1 versus 3, P<0.001), and lower Allred scores for estrogen and progesterone receptors (P = 0.002, 0.008). High Ki67 indices were also significantly correlated with higher Oncotype DX risk-of-recurrence group (low versus high, P<0.001). Ki67 index was the major contributor to a machine learning model which, when trained solely on clinico-pathological data and Ki67 scores, identified Oncotype DX high- and low-risk patients with 97% accuracy, 98% sensitivity and 80% specificity. Automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy, in a manner that integrates readily into the workflow of a pathology laboratory. Furthermore, automated Ki67 scores contribute significantly to models that predict risk of recurrence in breast cancer. [ABSTRACT FROM AUTHOR]

Mitotic count, PhH3, and MIB-1 are used as measures of the proportion of proliferating malignant cells in surgical pathology. They highlight different stages of the cell cycle, but little is known about how this affects their counts. This study assesses the strength of their correlations and attempts to determine the relationship between them. Proliferation counts for forty-nine consecutive cases of invasive breast carcinomas were analyzed, with the same tumor area on each stain counted using digital image analysis. The integrated optical density (IOD) of nuclei was measured as an approximation of nuclear DNA content. PhH3 strongly correlated with mitotic count (r = 0.94). Weaker correlations were found between MIB-1 versus PhH3 (r=0.79) and mitotic count (r=0.83). Nuclear IOD showed stronger correlation with MIB-1 (r=0.37) than to mitotic count (r=0.23) and PhH3 (r=0.34). With evidence from a literature review, it is suggested that the weaker correlations with MIB-1 are not explained by count imprecision or error, but relies on temporal decorrelation between cell cycle phases. Consequences on correlation between these proliferative markers are illustrated by mathematical models. [ABSTRACT FROM AUTHOR]

Background: Sox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood.Methods: In-vitro invasion assay was used to evaluate whether the expression of Sox2 is linked to the invasiveness of MCF7 and ZR751 cells. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and/or Western blots were used to assess if Sox2 modulates the expression of factors known to regulate epithelial mesenchymal transition (EMT), such as Twist1. Chromatin immunoprecipitation (ChIP) was used to assess the binding of Sox2 to the promoter region of Twist1.Results: We found that siRNA knockdown of Sox2 expression significantly increased the invasiveness of MCF7 and ZR751 cells. However, when MCF7 cells were separated into two distinct subsets based on their differential responsiveness to the Sox2 reporter, the Sox2-mediated effects on invasiveness was observed only in 'reporter un-responsive' cells (RU cells) but not 'reporter responsive' cells (RR cells). Correlating with these findings, siRNA knockdown of Sox2 in RU cells, but not RR cells, dramatically increased the expression of Twist1. Accordingly, using ChIP, we found evidence that Sox2 binds to the promoter region of Twist1 in RU cells only. Lastly, siRNA knockdown of Twist1 largely abrogated the regulatory effect of Sox2 on the invasiveness in RU cells, suggesting that the observed Sox2-mediated effects are Twist1-dependent.Conclusion: Sox2 regulates the invasiveness of BC cells via a mechanism that is dependent on Twist1 and the transcriptional status of Sox2. Our results have further highlighted a new level of biological complexity and heterogeneity of BC cells that may carry significant clinical implications. [ABSTRACT FROM AUTHOR]

Germ cell tumor with somatic malignant transformation is an uncommon phenomenon occurring about 7% of all mediastinal teratomas. Among all transformed component, sarcoma appears to be the most frequent histology, followed by primitive neuroectodermal tumor (PNET) and adenocarcinoma. To our knowledge, there were 3 cases of colonic-type adenocarcinoma arising in a primary teratoma have been reported to date. However, none of them received chemotherapy directed to transformed histology given localized disease at presentation. We, therefore, report here the first case of patient who achieved good response from chemotherapy directed to transformed histology, which confirms the importance of chemotherapy regimen used. [ABSTRACT FROM AUTHOR]

The author presents insights on the use of MYC immunohistochemistry assessment in diagnosing high-grade lymphomas. He states that 10 to 15% of diffuse large cell B-Cell lymphomas (DLBCL) are MYC driven. He cites a study which describes an IHC method for identifying DLBCL with increased MYC protein expression.

The article presents a case of a 71 year old woman who developed non‐purpuric cutaneous leukocytoclastic vasculitis after taking Tamoxifen for breast cancer. Topics discussed include Tamoxifen being an estrogen receptor modulator which is used as a first line adjuvant treatment in hormone receptor positive breast cancer.

Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. However, current approaches that rely on bright-field microscopy require extensive tissue preparation prior to imaging. These processes are both labor intensive and contribute to creating significant delays in clinical feedback for treatment decisions that can extend to 2–3 weeks for standard paraffin-embedded tissue preparation and interpretation, especially if ancillary testing is needed. Here, we present the first comprehensive study on the broad application of a novel label-free reflection-mode imaging modality known as photoacoustic remote sensing (PARS) for visualizing salient subcellular structures from various common histopathological tissue preparations and for use in unprocessed freshly resected tissues. The PARS modality permits non-contact visualizations of intrinsic endogenous optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. The technique was examined both as a rapid assessment tool that is capable of managing large samples (> 1 cm2) in under 10 min, and as a high contrast imaging modality capable of extracting specific biological contrast to simulate conventional histological stains such as hematoxylin and eosin (H&E). The capabilities of the proposed method are demonstrated in a variety of human tissue preparations including formalin-fixed paraffin-embedded tissue blocks and unstained slides sectioned from these blocks, including normal and neoplastic human brain, and breast epithelium involved with breast cancer. Similarly, PARS images of human skin prepared by frozen section clearly demonstrated basal cell carcinoma and normal human skin tissue. Finally, we imaged unprocessed murine kidney and achieved histologically relevant subcellular morphology in fresh tissue. This represents a vital step towards an effective real-time clinical microscope that overcomes the limitations of standard histopathologic tissue preparations and enables real-time pathology assessment. [ABSTRACT FROM AUTHOR]

Urothelial carcinoma (UC) is the most common form of bladder cancer. Although the 5-year overall survival rate of bladder cancer is 73%, the outcome for patients with metastatic UC remains poor. Programmed death ligand 1 (PD-L1) is over-expressed by many cancers and facilitates tumour evasion of host immune responses via the PDL1/PD-1 checkpoint. Immunotherapy is an emerging paradigm for the treatment of advanced UC with recent clinical trials demonstrating significant responses in patients by blocking PD-1/PD-L1. Nonetheless, the data on the prevalence of PD-L1 in urothelial tumours is limited. The goal of this study was to evaluate the prevalence of PD-L1 expression in tumour-infiltrating immune cells (IC) in UC in Canada and to correlate its expression with multiple clinical and pathological parameters. Three hundred archived UC specimens from six Canadian sites were assessed for PD-L1 expression using the Ventana PD-L1 (SP142) immunohistochemistry assay. The presence of discernible PD-L1 signal in tumour-infiltrating IC involving >5% of tumour with associated stroma was deemed "positive" and was observed in 39% (CI 33.13-44.43%) of specimens tested. There was no relationship between positivity of the PD-L1 assay and several clinicopathological parameters, including age, gender, and tumour location. Notably, PD-L1 expression of tumourinfiltrating IC exhibited a significant association with tumour stage (p=0.0409), with a trend towards higher PDL1 positivity in more advanced tumours. In conclusion, PD-L1 expression in tumour-infiltrating IC is common in UC in Canada and may be associated with more advanced stage cancers, reinforcing that PD-L1 may be a relevant therapeutic target for UC. [ABSTRACT FROM AUTHOR]

Surgical oncologists depend heavily on visual field acuity during cancer resection surgeries for in-situ margin assessment. Clinicians must wait up to two weeks for results from a pathology lab to confirm a post-operative diagnosis, potentially resulting in subsequent treatments. Currently, there are no clinical tools that can visualize diagnostically pertinent tissue information in-situ. Here, we present the first microscopy capable of non-contact label-free visualization of human cellular morphology in a reflection-mode apparatus. This is possible with the recently reported imaging modality called photoacoustic remote sensing microscopy which enables non-contact detection of optical absorption contrast. By taking advantage of the 266-nanometer optical absorption peak of DNA, photoacoustic remote sensing is efficacious in recovering qualitatively similar nuclear information in comparison to that provided by the hematoxylin stain in the gold-standard hematoxylin and eosin (H&E) prepared samples. A photoacoustic remote sensing system was employed utilizing a 266-nanometer pulsed excitation beam to induce photoacoustic pressures within the sample resulting in refractive index modulation of the optical absorber. A 1310-nanometer continuous-wave interrogation beam detects these perturbed regions as back reflected intensity variations due to the changes in the local optical properties. Using this technique, clinically useful histologic images of human tissue samples including breast cancer (invasive ductal carcinoma), tonsil, gastrointestinal, and pancreatic tissue images were formed. These were qualitatively comparable to standard H&E prepared samples. [ABSTRACT FROM AUTHOR]

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that is associated with significantly high mortality. In spite of advances in IBC diagnoses, the prognosis is still poor compared to non-IBC. Due to the aggressive nature of the disease, we hypothesize that elevated levels of inflammatory mediators may drive tumorigenesis and metastasis in IBC patients. Utilizing IBC cell models and patient tumor samples, we can detect elevated NF-κB activity and hyperactivation of non-canonical drivers of NF-κB (nuclear factor kappaB)-directed inflammation such as tyrosine phosphorylated receptor-interacting protein kinase 2 (pY RIPK2), when compared to non-IBC cells or patients. Interestingly, elevated RIPK2 activity levels were present in a majority of pre-chemotherapy samples from IBC patients at the time of diagnosis to suggest that patients at diagnosis had molecular activation of NF-κB via RIPK2, a phenomenon we define as “molecular inflammation”. Surprisingly, chemotherapy did cause a significant increase in RIPK2 activity and thus molecular inflammation suggesting that chemotherapy does not resolve the molecular activation of NF-κB via RIPK2. This would impact on the metastatic potential of IBC cells. Indeed, we can demonstrate that RIPK2 activity correlated with advanced tumor, metastasis, and group stage as well as body mass index (BMI) to indicate that RIPK2 might be a useful prognostic marker for IBC and advanced stage breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: Aberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets. Methods: We used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples. Results: We found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples. Conclusions: In conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences. [ABSTRACT FROM AUTHOR]