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There is an unmet clinical need for stratification of breast lesions as indolent or aggressive to tailor treatment. Here, single-cell transcriptomics and multiparametric imaging applied to a mouse model of breast cancer reveals that the aggressive tumor niche is characterized by an expanded basal-like population, specialization of tumor subpopulations, and mixed-lineage tumor cells potentially serving as a transition state between luminal and basal phenotypes. Despite vast tumor cell-intrinsic differences, aggressive and indolent tumor cells are functionally indistinguishable once isolated from their local niche, suggesting a role for non-tumor collaborators in determining aggressiveness. Aggressive lesions harbor fewer total but more suppressed-like T cells, and elevated tumor-promoting neutrophils and IL-17 signaling, disruption of which increase tumor latency and reduce the number of aggressive lesions. Our study provides insight into tumor-immune features distinguishing indolent from aggressive lesions, identifies heterogeneous populations comprising these lesions, and supports a role for IL-17 signaling in aggressive progression., The classification of breast lesions as indolent or aggressive to tailor treatment is crucial. Here, the authors use single-cell transcriptomics and multiparametric imaging of a breast cancer mouse model, report distinct tumor-immune features for the two types of lesions, and suggest the role of IL-17 signaling in disease progression.

Ductal carcinoma in situ (DCIS) is a non-invasive proliferative growth in the breast that serves as a non-obligate precursor to invasive ductal carcinoma. The widespread adoption of screening mammography has led to a steep increase in the detection of DCIS, which now comprises approximately 20% of new breast cancer diagnoses in the United States. Interestingly, the intratumoral heterogeneity (ITH) that has been observed in invasive breast cancers may have been established early in tumorigenesis, given the vast and varied ITH that has been detected in DCIS. This review will discuss the intratumoral heterogeneity of DCIS, focusing on the phenotypic and genomic heterogeneity of tumor cells, as well as the compositional heterogeneity of the tumor microenvironment. In addition, we will assess the spatial heterogeneity that is now being appreciated in these lesions, and summarize new approaches to evaluate heterogeneity of tumor and stromal cells in the context of their spatial organization. Importantly, we will discuss how a growing understanding of ITH has led to a more holistic appreciation of the complex biology of DCIS, specifically its evolution and natural history. Finally, we will consider ways in which our knowledge of DCIS ITH might be translated in the future to guide clinical care for DCIS patients.

Objective To describe the rapid conceptualization and implementation of an interdisciplinary epilepsy clinic for children with drug-resistant epilepsy (DRE) at Arkansas Children's Hospital (ACH) during the COVID 19 pandemic. Methods Focusing on care design and care coordination for children with DRE, multiple stakeholder groups decided to implement a clinic after the systematic rating of constructs present in a theoretical meta-analytic framework. Based on the projected success, the new interdisciplinary clinic (composed of an epileptologist, a neurosurgeon, and a neuropsychologist and coordinated by a full-time nurse) was established. Clinic operations were further refined through discussions with patients, families, and care providers. We collected data retrospectively (August 2020 to June 2021) to determine referral patterns, clinic scheduling metrics, patient characteristics, clinical recommendations, and epilepsy quality metrics. Results Of the 32 Consolidated Framework for Implementation Research constructs assessed, 24 were positively rated to predict a high probability of successful implementation of the clinic. For approximately 100 patient visits, appearance and usage rates were >75%, yielding a clinic utilization rate of approximately 60%. Among 76 unique patients (average age of 12 years, 60% focal epilepsy), 39 patients (51.3%) were deemed eligible for epilepsy surgery evaluation. The majority of the patients (53.9%) were advised for additional diagnostic testing, and 31.6% of patients were scheduled for vagus nerve stimulation. More patients (33%) had changes in their existing anti-seizure medication (ASM) regimen rather than an addition of a new ASM (7.9%). Standardized epilepsy quality measures showed >80% to 90% adherence in 3 (reproductive counseling, depression and anxiety screening, documentation of seizure frequency) out of 4 metrics. Significance This is the first study to show that an interdisciplinary clinic can be a valuable attribute of care models in high-need children with DRE by enabling comprehensive one-stop service for diagnostic evaluation, surgical consideration, and brief assessment of psychiatric comorbidities without compromising consensus-based best practices.

The relationship between ATR/Chk1 activity and replication stress, coupled with the development of potent and tolerable inhibitors of this pathway, has led to the clinical exploration of ATR and Chk1 inhibitors (ATRi/Chk1i) as anticancer therapies for single-agent or combinatorial application. The clinical efficacy of these therapies relies on the ability to ascertain which patient populations are most likely to benefit, so there is intense interest in identifying predictive biomarkers of response. To comprehensively evaluate the components that modulate cancer cell sensitivity to replication stress induced by Chk1i, we performed a synthetic-lethal drop-out screen in a cell line derived from a patient with triple-negative breast cancer (TNBC), using a pooled barcoded shRNA library targeting ~350 genes involved in DNA replication, DNA damage repair, and cycle progression. In addition, we sought to compare the relative requirement of these genes when DNA fidelity is challenged by clinically relevant anticancer breast cancer drugs, including cisplatin and PARP1/2 inhibitors, that have different mechanisms of action. This global comparison is critical for understanding not only which agents should be used together for combinatorial therapies in breast cancer patients, but also the genetic context in which these therapies will be most effective, and when a single-agent therapy will be sufficient to provide maximum therapeutic benefit to the patient. We identified unique potentiators of response to ATRi/Chk1i and describe a new role for components of the cytosolic iron–sulfur assembly (CIA) pathway, MMS19 and CIA2B-FAM96B, in replication stress tolerance of TNBC.

Shifts in tumor phenotype in response to selective pressures (i.e. changing microenvironments, drug treatments) pose one of the biggest obstacles to successful cancer therapies. Phenotypically diverse tumor and stroma subpopulations, and interactions between them that alter tumor biology, represent unique signaling niches. We hypothesize that these localized neighborhoods of breast tumor cells possess specialized phenotypes that mediate chemoresistance and represent novel therapeutic vulnerabilities. In order to assess these potential phenotypes, we utilized imaging mass cytometry (IMC), a highly multiplexed imaging modality that allows simultaneous measurement of 30-40 antigens while retaining the spatial architecture of the cancer tissue. We have constructed an IMC antibody panel that combines markers for tissue architecture, tumor and stromal cell phenotyping, and signaling pathway activation. IMC was applied to a collection of patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC). Our PDX collection was established from patient tumors obtained before and after neoadjuvant chemotherapy. IMC analysis of 18 PDX models representing eight patients revealed that stromal cell phenotypes were generally shared between all models, but tumor cell phenotypes were largely patient-specific. While every model was comprised primarily of a few major tumor cell phenotypes, we noted that each case also harbored several minor, unique populations, suggesting that specialized neighborhoods may exist within the tumor mass. Sequential PDX models showed a wide range of phenotypic responses after chemotherapy, ranging from stable tumor composition to widespread changes in tumor phenotypes. Importantly, comparison of multiple pre-/post-chemotherapy PDX pairs revealed spatially constrained MAPK activation emerging after treatment. To capture acute changes in tumor phenotype, we treated animals bearing PDX tumors with chemotherapy and evaluated tumors by IMC. As tumors regressed and then regrew, we identified novel phenotypic shifts, again including increased MAPK signaling localized to discrete neighborhoods, suggesting this phenomenon may be a common feature of chemoresistant TNBC. Taken together, our findings suggest that distinct tumor phenotypes arise following treatment. Ongoing work is examining the spatial arrangement of these cell types to determine their local niche compositions, and then correlating these findings with clinical features. Our goal is to determine whether these unique signaling niches functionally contribute to chemoresistance and if disruption of these niches enhances chemosensitivity. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Jiansu Shao, Xiaomei Zhang, Gloria V. Echeverria, W. Fraser Symmans, Stacy L. Moulder, Helen Piwnica-Worms. Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR01.

Introduction: Ductal carcinoma in situ is a non-invasive lesion of the breast that comprises ~20% of newly diagnosed breast cancers in the United States. Prospectively distinguishing indolent from aggressive lesions has been a major clinical challenge. To begin addressing these challenges, we use an experimentally tractable mouse model of breast cancer in which invasive (but rarely in situ) lesions display architectural and morphological abnormalities while also being heavily immune-infiltrated. Methods: To explore the tumor-stroma niches that evolve as breast cancer progresses, we are undertaking single cell RNA sequencing and imaging mass cytometry to identify tumor and immune cells present within in situ (early) versus invasive (advanced) lesions, and to characterize shifts in phenotype and tumor-stroma niches that may occur as breast cancer transitions from in situ to invasive disease. Results: Single cell analyses revealed major cell populations within the tumor compartment of both early and advanced disease, including luminal- and basal-like populations, as well as an additional minor population that appears to represent a unique intermediate between luminal and basal states. Early stage lesions were comprised primarily of the major luminal population, whereas advanced lesions exhibited a significant expansion of intermediate and basal populations, suggesting that breast cancer cells may undergo a transition from luminal-like to basal-like phenotype during progression to invasive disease. Analysis of the immune compartments of early versus advanced disease also revealed that the relative neutrophil/MDSC/S100A8+ population increased as lesions advanced, while B cells, NK cells, and some T cells decreased. Furthermore, the immune populations of advanced lesions exhibited widespread upregulation of IL-17 targets, suggesting that the IL-17 pathway may drive shifts in the immune microenvironmental to support disease progression, particularly the increase in neutrophils/MDSCs. Additionally, given a previously reported role of IL-17 to alter epithelial stemness, increased IL-17 signaling in advanced lesions may also be driving the potential luminal-to-basal transition of malignant cells. Conclusion: Taken together, these findings suggest that a subset of malignant epithelial cells may transition from luminal-like to basal-like cells, and that this transition may be driven and/or maintained by a commensurate increase in IL-17 signaling that both directly impacts tumor cells and also establishes a tumor-supportive microenvironment. Citation Format: Vidya C. Sinha, Mingchu Xu, Amanda L. Rinkenbaugh, Xinhui Zhou, Xiaomei Zhang, Helen Piwnica-Worms. Single-cell evaluation to identify tumor-stroma niches driving the transition from in situ to invasive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2822.

In response to oncogene activation and oncogene-induced aberrant proliferation, mammalian cells activate apoptosis and senescence, usually via the p53–ARF tumor-suppressor pathway. Apoptosis is a known barrier to cancer and is usually downregulated before full malignancy, but senescence as an anticancer barrier is controversial due to its presence in the tumor environment. In addition, senescence may aid cancer progression via releasing senescence-associated factors that instigate neighboring tumor cells. Here, it is demonstrated that apoptosis unexpectedly remains robust in ErbB2 (ERBB2/HER2)-initiated mammary early lesions arising in adult mice null for either p53 or ARF. These early lesions, however, downregulate senescence significantly. This diminished senescence response is associated with accelerated progression to cancer in ARF-null mice compared with ARF–wild-type mice. Thus, the ARF–p53 pathway is dispensable for the apoptosis anticancer barrier in the initiation of ErbB2 breast cancer, the apoptosis barrier alone cannot halt mammary tumorigenesis, and senescence is a key barrier against carcinogenesis. Implications: Findings in this relevant mouse model of HER2-driven breast cancer suggest that effective prevention relies upon preserving both ARF/p53–independent apoptosis and ARF/p53–dependent senescence. Mol Cancer Res; 13(2); 231–8. ©2014 AACR.

Lung Cancer is a disease can be defined as uncontrolled cell growth in tissues of the lung. If we detect the Lung Cancer in its early stage, then that could be the key of its cure. In this work the non-invasive methods are studied for assisting in nodule detection. It supplies a Computer Aided Diagnosis System (CAD) for early detection of lung cancer nodules from the Computer Tomography (CT) images. CAD system is the one which helps to improve the diagnostic performance of radiologists in their image interpretations. The main aim of this technique is to develop a CAD system for finding the lung cancer using the lung CT images and classify the nodule as Benign or Malignant. For classifying cancer cells, SVM classifier is used. Here, image processing techniques have been used to de-noise, to enhance, for segmentation and edge detection of an image is used to extract the area, perimeter and shape of nodule. The core factors of this research are Image quality and accuracy.

Background: Liver being the largest organ in the body receives blood supply from hepatic artery (25%) and portal vein (75%) and drained by three major hepatic veins opening into inferior vena cava. The dimensions of hepatic veins are quite essential during portosystemic shunting procedure and radiological interventions. The presence of variants of major hepatic veins is of prime importance to evaluate donor and recipient in living donor liver transplantation. Objectives: The objectives of the study were (1) to evaluate the ramification pattern and morphometry of major hepatic veins of liver by cast method and (2) to study the clinical implications of the hepatic vein variations. Materials and Methods: Forty liver specimens (22 males and 18 females) aged between 30 and 60 years were obtained from the Department of Forensic Medicine of JSS Medical College and Mysore Medical College. The silicone gel was injected into vena cava and pushed thoroughly into the ostia of hepatic veins. The drainage patterns of hepatic veins were noted and measurement of hepatic veins was done using thread and measuring scale. Results: The present study ramification pattern of middle and left hepatic veins showed Type 1 pattern in 24 specimens (58%), Type 2 in 12 specimens (40%), and Type 3 in 5 specimens (12%). There was a significant difference with P < 0.05 in all the parameters among males and females except the length and diameter of common trunk. Conclusion: Thorough knowledge of hepatic vein variants and its morphometry is essential during imaging procedures, anatomic resection of hepatic veins in hepatocellular carcinoma, liver transplantation, and other liver surgeries.

Tumors are highly heterogeneous populations of cells, and measures of intratumoral heterogeneity (ITH) and diversity correlate with worse prognosis in many cancers, including breast cancer. Emerging studies are highlighting functional interactions between subclones, as well as among subclones and components of the tumor microenvironment. However, these studies have largely focused on soluble factors without interrogating the spatial distribution of subclones defined by activated signaling pathways. Previous work in this area has been severely limited by technical restrictions - existing techniques allowing measurement of many biomarkers simultaneously lose all information about the tissue architecture, while those that do retain spatial information can only assay a handful of markers at once. We will circumvent these limitations by undertaking imaging mass cytometry (IMC), which allows for simultaneous measurement of 30-40 antigens while retaining the spatial organization of the sample. Our objective is to dissect the signaling heterogeneity in tumors from patients and patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), through two main approaches: (1) characterization of signaling heterogeneity in tissue microarrays of human tumors and PDX models of TNBC with IMC and (2) modeling cell signaling heterogeneity in cell line-based models to determine mechanisms of cell-cell interaction and communication. We have constructed an IMC panel of antibodies that combines markers for tissue architecture, tumor and immune cell phenotyping, and signaling pathway activation. Profiling of a diverse panel of TNBC PDX models captures the heterogeneity of human TNBC. Analysis of distinct regions within individual PDX tumors demonstrate unique compositions of cell phenotypes between the edge and core of the tumor. Our PDX collection also includes sequential pairs derived from biopsies taken before and after chemotherapy treatment. Comparison of the pre-/post-chemotherapy pairs indicates emerging patterns of pathway activation. Interestingly, while the tumor cells from these models exhibit distinct phenotypes, the stromal cells are largely indistinguishable from one another, suggesting that these models are capturing tumor cell-intrinsic changes associated with chemotherapy response. Ultimately, we plan to use these results to identify novel vulnerabilities in subclonal interactions that can be targeted therapeutically in TNBC. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Xiaomei Zhang, Jiansu Shao, Helen Piwnica-Worms. Investigating triple negative breast cancer phenotypic heterogeneity of human and patient-derived xenograft samples using imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4708.

Our overall goal is to distinguish aggressive from indolent breast cancers by characterizing determinants of breast cancer progression. The detection of early-stage (in situ) neoplastic lesions in the breast has risen dramatically due to widespread mammography. Early detection represents a significant opportunity to intervene and halt the progression of these early lesions to invasive breast cancer. However, our understanding of the natural history of in situ lesions remains limited. Here, we describe our efforts to characterize the microenvironment of DCIS in patient samples and mouse models using a combination of traditional and novel multiplexed immunostaining methods. In particular, we present our early work on applying multispectral fluorescence imaging as well as highly parametric imaging mass cytometry to describe the immune cells surrounding and infiltrating DCIS, with focus on the interaction of these immune cells with each other and the incipient invasive tumor cells. Future studies will be directed at determining if the immune microenvironment of DCIS can identify patients at risk for progression or recurrence so that these patients can be appropriately managed upfront. Citation Format: Vidya C. Sinha, Fei Yang, Amanda L. Rinkenbaugh, Xinhui Zhou, Xiaomei Zhang, Ignacio I. Wistuba, Helen Piwnica-Worms. Multiparametric imaging to evaluate human and mouse breast carcinoma in situ [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B59.

Tumors are highly heterogeneous populations of cells, and measures of intratumoral heterogeneity (ITH) and diversity correlate with worse prognosis in many cancers, including breast cancer. Emerging studies are highlighting functional interactions between subclones, as well as among subclones and components of the tumor microenvironment. However, these studies have largely focused on soluble factors without interrogating the spatial distribution of subclones defined by activated signaling pathways. Previous work in this area has been severely limited by technical restrictions – existing techniques allowing measurement of many biomarkers simultaneously lose all information about the tissue architecture, while those that do retain spatial information can only assay a handful of markers at once. We will circumvent these limitations by undertaking imaging mass cytometry (IMC), which allows for simultaneous measurement of 30-40 antigens while retaining the spatial organization of the sample. Our objective is to dissect the signaling heterogeneity in tumors from patients and patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), through two main approaches: (1) characterization of signaling heterogeneity in human tumors and PDX models of TNBC through the use of imaging mass cytometry (IMC) and (2) modeling cell signaling heterogeneity in cell line-based models to determine mechanisms of cell-cell interaction and communication. We have constructed an IMC panel of antibodies that combines markers for tissue architecture, cell phenotyping, and signaling pathway activation. Profiling of a panel of TNBC PDX models, including matched sets of pre- and post-chemotherapy treatment, indicates emerging patterns of pathway activation. We will utilize these results to inform functional studies in our isogenic cell line models. As it is becoming more clear that tumors are composed of heterogeneous subclones, it is crucial that we understand the communication between subclones and with components of the microenvironment. Ultimately, we plan to use these results to identify novel vulnerabilities in subclonal interactions that can be targeted therapeutically in TNBC. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Xiaomei Zhang, Helen Piwnica-Worms. Functionalizing intratumoral signaling heterogeneity in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2181.

Glucose transporters, such as GLUT-1, mediate the important mechanisms involved in cellular glucose influx, allowing cells to proliferate and survive. The significance of GLUT-1 expression in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) has been less explored, and no study has investigated it in relation to verrucous carcinoma (VC). We evaluated 30 cases each of OED, OSCC, and VC, graded further on the basis of their differentiation, immunohistochemically for GLUT-1 expression, along with 10 specimens of normal oral mucosa (NOM) as controls. In OSCC, GLUT-1 expression increased with the degree of dysplasia and increasing grade (P < 0.001). The expression in VC was predominantly membranous and intense, resembling well differentiated OSCC. This increase of GLUT-1 expression in OSCC along with the degree of dysplasia and the histologic grade reflects the expanding glycolytic response to hypoxia. This is the first study to have revealed prominent GLUT-1 expression in VC, highlighting its inherent metabolic capacity.