Stromal Regions Research Articles

Abstract 6575: A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity

Abstract Spatial genomics and digital pathology continue to advance at a rapid pace, generating large amounts of data. There is a critical unmet need for advanced tools to expedite spatial data generation, visualization, and analysis, to facilitate an improved understanding of health and disease, especially with the advancement of personalized cancer treatments. In this study we present a novel method for the extraction and analysis of genomic information from specific regions of interest (ROI) within a series of formalin-fixed paraffin embedded (FFPE) breast cancer adenocarcinoma tissue sections. An artificial intelligence-based tumor profiling algorithm first identified tumor, stroma, inflammatory, and necrotic regions within the tissue, and segmentation results were confirmed by a Board Certified Pathologist. Automated lysate extraction was then performed in the selected ROIs using ink-jet technology and novel ink chemistry. The process was performed directly on the tissue slide and the crude lysates generated were transferred to a standard collection tube for subsequent sequencing. DNA was extracted from the selected areas before being purified and analyzed using a multi-cancer panel. Library QC showed optimal coverage at an average of >95% uniformity. Sequencing results showed an average coverage of 95% at 250x across all amplicons of the panel. Variant calling was successfully performed. Sequencing data is presented to demonstrate the high coverage achieved using this workflow as well as the resulting tumor heterogeneity. This novel technology provides an efficient solution for the enrichment of tumor content with high sequencing coverage from FFPE tissue sections. Novel spatial data visualization and analysis techniques allowed a comprehensive assessment of tumor heterogeneity. Further studies on both DNA and RNA should be conducted to validate this approach for clinical molecular testing. Citation Format: Bidhan Chaudhuri, Katie Konigsfeld, Marisa Sanchez, Jason Smith, Casey Laris, Claire Weston, John Butler. A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity [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 6575.

Abstract 5105: Unravelling the mystery of cancer-associated fibroblast populations in the tumor microenvironment by a fully automated sequential chromogenic multiplex assay

Abstract The tumor microenvironment (TME) is one of the driving factors of tumor progression and invasion. Inside this microenvironment, Cancer-Associated Fibroblasts (CAF) play a significant role in tumor modulation and in the development of drug resistance. This major stromal cell population has been highlighted as a significant prognostic factor in several tumors. CAF are characterized by an original, phenotypic and function heterogeneity, as well as a high plasticity. Several studies have demonstrated that this population contains different subtypes with the capacity to promote or inhibit cancer. Moreover, their biological role as allies or foes is dependent on many factors, including the cancer stage. There is yet no consensus on the molecular definition of these key TME players, but several markers have been used to identify them. However, many of these markers come with their own set of downsides, such as low specificity and inability to detect different subtypes within the wider CAF population. In this context, novel refined immunophenotyping approaches such as multiplex technology are vital for accurate identification and quantification of CAF. Here we assessed the presence, abundance and localization of CAF subsets within the pancreatic cancer microenvironment by using the Brightplex® solution, an automated sequential chromogenic multiplex assay. A unique combination of biomarkers (Fibroblast activation protein (FAP), Alpha smooth muscle actin (αSMA), Platelet derived growth factor receptor alpha/beta (PDGFRα/β), Fibroblast specific protein 1 (FSP-1), negative markers) was developed to assess CAF heterogeneity and plasticity on a single FFPE tumor tissue section by preserving tissue's spatial contexture. Briefly, a tissue section was sequentially stained, digitized, unstained and re-stained with antibodies targeting the selected markers. Images of the whole slide were then analyzed by digital pathology and the detection of positive cells was performed for each marker independently. In addition, tissue segmentation tools were used to assess CAF densities in parenchyma, tumor stroma and invasive margin regions. This precise characterization of CAF subtypes has been performed on pancreatic ductal adenocarcinoma FFPE tissues. The several subtypes of CAF were resolved spatially and studied in relation to CD3+ and CD8+ T cell infiltration (Immunoscore®) at different stages of pancreatic cancer development. This new tool allows to decipher the heterogeneity and plasticity of CAF to evaluate their contribution to disease progression and help clinical researchers to understand mode of action of CAF-targeting drugs. This new Brightplex® panel will be integrated into our Immunogram to better understand the immune contexture of the tumor. Citation Format: Thomas Sbarrato, Mounia Filahi, Véronique Frayssinet, Anne-Laure Bailly, Caroline Laugé, Caroline Davin, Hélène Girardi, Anna Martirosyan, jacques Fieschi. Unravelling the mystery of cancer-associated fibroblast populations in the tumor microenvironment by a fully automated sequential chromogenic multiplex assay [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 5105.

Abstract 2677: Sequential same slide multiplex immunofluorescence and H&E staining for combined phenotypic and morphologic characterization of formalin-fixed paraffin-embedded tissue sections

Abstract Multiplex immunofluorescence (mIF) has become an established method for characterizing the tumor immune microenvironment. Currently, hematoxylin and eosin (H&E) staining is typically performed on a serial tissue section to provide morphologic context for the mIF data. Serial sections do not represent identical cell populations, however, and are not always possible to obtain when the amount of available tissue is limited. Consequently, a method to examine the same tissue section by both fluorescence and brightfield imaging would be advantageous. Here we present a streamlined method and system for serial mIF and H&E staining on a single tissue slide for a comprehensive analysis to support high-throughput tissue immunophenotyping. The UltiMapper I/O PD-L1 kit was used for multiplex immunofluorescence staining of CD8, CD68, PD-L1, and pan-Cytokeratin in formalin-fixed, paraffin-embedded (FFPE) samples from human tonsil and primary tumor biopsies using the Leica Biosystems BOND RX autostainer. Stained tissues were imaged in five spectrally distinct fluorescence channels (DAPI, FITC, TRITC, Cy5, Cy7) on the RareCyte CyteFinder II HT Instrument. Slides were de-coverslipped and stained with H&E, then imaged with brightfield using the CyteFinder II HT instrument. To segment the tumor and stroma tissue regions, a HALO AI classifier was created. Fluorescence images were analyzed using the HALO Highplex FL module to identify CD8+ cytotoxic T-cells, CD68+ macrophages, CD68+/PD-L1+ immuno-suppressive macrophages, pan-CK+ tumor cells, and pan-CK+/PD-L1+ immune-evading tumor cells within the tumor and stromal regions identified by the H&E stain. This novel combination of fluorescence and brightfield analysis enables deep phenotyping of immune cells as well as assessment of cellular morphology and tumor architecture in the same tissue section. Citation Format: Katir K. Patel, Amanda Bares, Mael Manesse, Mark Burton, Bonnie Phillips, Kate Lillard, Anne Hellebust, Melinda Duplessis, Kyla Teplitz, Tad George. Sequential same slide multiplex immunofluorescence and H&E staining for combined phenotypic and morphologic characterization of formalin-fixed paraffin-embedded tissue sections [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 2677.

Characterization of colorectal mucus using infrared spectroscopy: a potential target for bowel cancer screening and diagnosis

AbstractBiological materials presenting early signs of cancer would be beneficial for cancer screening/diagnosis. In this respect, the suitability of potentially exploiting mucus in colorectal cancer was tested using infrared spectroscopy in combination with statistical modeling. Twenty-six paraffinized colon tissue biopsy sections containing mucus regions from 20 individuals (10 normal and 16 cancerous) were measured using mid-infrared spectroscopic imaging. A digital de-paraffinization, followed by cluster analysis driven digital color-coded multi-staining segmented the infrared images into various histopathological features such as epithelium, connective tissue, stroma, and mucus regions within the tissue sections. Principal component analysis followed by supervised linear discriminant analysis was carried out on pure mucus and epithelial spectra from normal and cancerous regions of the tissue. For the mucus-based classification, a sensitivity of 96%, a specificity of 83%, and an area under the curve performance of 95% was obtained. For the epithelial tissue-based classification, a sensitivity of 72%, a specificity of 88%, and an area under the curve performance of 89% was obtained. The mucus spectral profiles further showed contributions indicative of glycans including that of sialic acid changes between these pathology groups. The study demonstrates that infrared spectroscopic analysis of mucus discriminates colorectal cancers with high sensitivity. This concept could be exploited to develop screening/diagnostic approaches complementary to histopathology.Mucus was tested as a potential biological material for screening/early diagnosis of colorectal cancer using infrared spectroscopy. Based on a digital histopathology and statistical modeling approach, cancerous and non-cancerous samples were classified with an area under the curve performance of 95% based on mucus spectral profiles, indicating changes in the glycan component of mucins.

Poly(ADP-ribose) polymerase-2 is essential for endometrial receptivity and blastocyst implantation, and regulated by caspase-8

Embryo implantation is a very complex process and several factors play important roles. Using a mouse model, we investigated the functions of PARP-2 and caspase-8 during endometrial receptivity for blastocyst implantation. We found that PARP-2 was upregulated at the receptive stage's implantation region and predominantly expressed in the endometrial stromal region, but downregulated during pregnancy failure and pseudopregnancy. To reinforce the necessity of PARP-2 for embryo implantation, we pharmacologically inhibited PARP-2 ‘before’ & ‘after’ embryo arrival and observed a reduction in blastocyst implantation. Conversely, elevated caspase-8 expression and activity during pseudopregnancy, delayed implantation, and embryo implantation failure conditions and decreased levels in the decidualization exhibited an inverse pattern with PARP-2, suggesting caspase-8 as a negative regulator for embryo implantation. In vitro caspase-8 downregulates the PARP-2 activity in the mouse endometrial epithelial and stromal cells. These data suggest that PARP-2 and its negative regulation by caspase-8 constitute a crucial step in embryo implantation.

Magnetic resonance diffusion tensor imaging of cervical microstructure in normal early and late pregnancy in vivo

Cervical remodeling is an important aspect of birth timing. Before cervical ripening, the collagen fibers are arranged in a closely interweaved network, but during ripening, the fibers become disorganized and the cervix becomes more hydrated. To quantitatively measure cervical remodeling, we need a noninvasive method to monitor changes in cervical collagen fiber organization and hydration invivo. To use diffusion tensor imaging to image and quantify the spatial and temporal differences in cervical microstructure between normal early and late pregnancies. After institutional review board approval and consent, a group of healthy women in early pregnancy (22 patients at 12-14 weeks' gestation) and a group in late pregnancy (27 patients at 36-38 weeks' gestation) underwent magnetic resonance imaging on a Siemens MAGNETOM Vida 3 Tesla unit. Diffusion tensor imaging of the cervix in the axial plane was performed with a two-dimensional single-shot echo planar imaging diffusion-weighted sequence. In early and late pregnancy groups, the differences of the diffusion tensor imaging measures were compared between the subglandular zone and the outer stroma regions of the cervix. In addition, the diffusion tensor imaging measures were compared between the early and late pregnancy groups. Finally, for the late pregnancy group, the diffusion tensor imaging measures were compared between the primipara and multipara groups. Diffusion tensor imaging measures of microstructure significantly differed between the subglandular zone and outer stroma regions of the cervix in both early and late pregnancies. In the subglandular zone, fractional anisotropy was lower in the late pregnancy group than in the early pregnancy group (0.37 [0.34-0.42] vs 0.50 [0.43-0.58]; P<.0005), suggesting increased collagen fiber disorganization in this zone. In addition, mean diffusivity was higher in the late pregnancy group than in the early pregnancy group (1.84 [1.73-2.02] mm2/sec×10-3 vs 1.56 [1.42-1.69] mm2/sec×10-3; P=.001), suggesting increased hydration in the subglandular zone. In the outer stroma, neither fractional anisotropy (0.44 [0.40-0.50] vs 0.41 [0.37-0.43]; P=.095) nor mean diffusivity (2.09 [1.92-2.25] mm2/sec×10-3 vs 2.12 [2.04-2.24] mm2/sec×10-3; P=.269) significantly differed between early pregnancy and late pregnancy, suggesting insignificant temporal microstructural changes in this cervical zone. Diffusion tensor imaging measures did not significantly differ between cervixes from primiparous and multiparous women in late pregnancy. This invivo study demonstrates that diffusion tensor imaging can noninvasively quantify the microstructural differences in collagen fiber organization and hydration in cervical subregions between early pregnancy and late pregnancy.

Abstract B76: Pyruvate dehydrogenase: A key to epigenetic regulation in CAFs

Abstract Cancer-associated fibroblasts (CAFs) play fundamental roles in cancer and are emerging as therapeutic target in tumors with extensive stromal regions and in those for which there are limited targeted therapies against the cancer cells, such as ovarian cancer. A unique feature of the CAFs is their ability to secrete abundant collagen-rich extracellular matrix (ECM) that promotes the desmoplastic reaction that accompanies tumor progression and drives tumor growth and metastasis. Altered tumor metabolism is a hallmark of cancer, and understanding whether and how metabolic pathways support protumorigenic and proinvasive CAF functions may identify ways to target these cells to effectively target tumors. Using global phosphoproteomics, we have found that the activity of the pyruvate dehydrogenase complex (PDC), which is the rate-limiting enzyme for the entry of glycolysis-derived metabolites into the TCA cycle by converting pyruvate into acetyl-CoA, is strongly increased in patient-derived CAFs compared to their normal fibroblast counterpart. Consistently, the expression of pyruvate dehydrogenase kinase (PDK), which phosphorylates and inhibits PDC activity, is downregulated in CAFs and in the stroma of tumor patient samples. We found that PDC activity in CAFs leads to increased acetyl-CoA production. Surprisingly, 13C-glucose tracing experiments showed that CAFs do not channel acetyl-CoA into the TCA cycle. Instead, CAFs use acetyl-CoA to activate an epigenetic switch triggered by acetylation of H3K27. H3K27 acetylation is a known marker of gene expression activation. In CAFs, it triggered the expression of several collagen genes. Interestingly, also the expression of enzymes of the proline synthesis pathway was induced following H3K27 acetylation. Collagens have an unusually high content of proline residues, and we show that enhanced proline synthesis is necessary to support the production of collagen-rich ECM in CAFs. Targeting the PDK/PDC pathway or H3K27 acetylation or the proline synthesis pathway was sufficient to inhibit collagen synthesis in CAFs in in vitro experiments. Targeting proline synthesis in the stroma was sufficient to reduce tumor growth in vivo. Our work provides a first evidence that metabolism and epigenetics are tightly intertwined in regulating CAF functions and that targeting the PDK/PDC pathway or the proline synthesis pathway in the stroma could halt the development of a desmoplastic reaction and tumor progression. Citation Format: Emily Kay, Lisa Neilson, Claudia Boldrini, Juan Hernandez-Fernaud, Enio Gjerga, David Sumpton, Sandeep Dhayade, Grace McGregor, Grigorios Koulouras, Jurre Kamphorst, Karen Blyth, Julio Saez-Rodriguez, Gillian Mackay, Sara Zanivan. Pyruvate dehydrogenase: A key to epigenetic regulation in CAFs [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B76.

Biomechanical effect of ultraviolet-A-riboflavin cross-linking on simulated human corneal stroma model and its correlation with changes in corneal stromal microstructure

In this study, we established an experimental human corneal stroma model of simulated cornea tissue composed of thin anterior cornea strips layers obtained from small incision lenticular extraction (SMILE) surgery. We investigated the biomechanical effect of ultraviolet-A- riboflavin cross-linking at different depths of corneal stroma model and correlated it with stromal microstructural changes examined by transmission electron microscopy (TEM). Corneal strips were harvested from fresh human corneal lenticules obtained after SMILE surgery. Experimental models (n = 34) were established by superimposing the corneal lenticule strips until their thickness reached close to 500 μm. Corneal cross-linking (CXL) was performed subsequently using standard or accelerated protocol. Elasticity and viscosity were quantified using stress-strain extensometer. TEM was used to visualize the collagen fiber diameter and interfibrillar spacing. The relative change in Young's modulus (rel. ΔE) decreased nonlinearly with increasing stromal depth both in the standard and accelerated groups. Compared to the sham controls, the rel. ΔE in standard and accelerated CXL groups increased significantly in the anterior 400 μm and 275 μm depth, respectively. Also, the relative change in stress (rel. ΔS) was significantly lower after standard and accelerated CXL compared to sham controls. Depth analysis showed similar results for the elastic effect. TEM images showed a small, non-significant increase in fibril diameter. The interfibrillar spacing decreased significantly after standard and accelerated CXL in the anterior-mid stromal region. We noted that the increase of corneal stiffness correlated with decrease in interfibrillar spacing after CXL. The stiffening effect was depth dependent. The effect of accelerated CXL was less in the deep corneal stromal regions compared to standard CXL.

The relationship between tumor-infiltrating lymphocytes, PD-L1 expression, and clinical outcomes of Asian melanoma patients treated with anti-PD-1 antibody.

e22010 Background: Clinical feature of Asian melanoma patients are distinct from those of Western patients. Acral and mucosal melanomas, which are common in Asian populations, predominantly occur in sun-protected areas. Immune-checkpoint inhibitors are standard treatment for inoperable melanoma, however, they are less effective against acral and mucosal melanomas. The aim of our study is to investigate the expression, clinical significance and association of programmed death-ligand 1 (PD-L1) with tumor-infiltrating lymphocytes (TIL) in Asian melanoma patient treated with anti-PD-1 therapy. Methods: Patients with unresectable stage III or metastatic stage IV melanoma who began anti-PD-1 antibody therapy between February 2016 and September 2018 were enrolled. Immunohistochemistry was used to evaluate PD-L1 expression and stromal CD8+ TIL, and PD-1+ TIL densities within the intratumoral regions and associated stromal regions. Baseline characteristics and blood parameters including neutrophil to lymphocyte ratio (NLR) were assessed, and outcome and adverse events were evaluated according to subtypes. Results: Of the 57 cases, the most common type was acral melanoma (n = 28, 49.1%) followed by mucosal melanoma (n = 19, 33.3%) and cutaneous (n = 10, 17.5%). The overall response was 8.8 % and disease control rate was 33.3%. The median progression-free survival (PFS) was 18.0 months (95% confidence interval, 12.57 to 23.43), and there was no difference in PFS according to subtypes. Overall, 29.8% (n = 17) showed PD-L1 expression in tumor cells and 26.3% in immune cells. PD-L1, CD8+TIL, LDH and BRAFV600 statues were not associated with response or survival. NLR (≥ 5) were independent poor prognostic factors by multivariate analysis. Conclusions: In our study, anti-PD-1 therapy showed less effective compared to western patients. High NLR (≥ 5) could predict response of anti-PD-1 therapy in patients with Asian melanoma. Considering the differences in genetic backgrounds and therapeutic efficacy of immunotherapy, specialized therapeutic strategies for Asian melanoma should be established.

PD-1 topographically defines distinct T cell subpopulations in urothelial cell carcinoma of the bladder and predicts patient survival

IntroductionThe pre-existing tumor-infiltrating T cell landscape in urothelial cell carcinoma of the bladder (UCB) may obtain prognostic significance and guide treatment decisions, particularly regarding immunotherapy. However, the current studies typically lead to inconsistent conclusions due to the extreme heterogeneity of T cells in cancer. Herein, we investigated the heterogeneity, distribution and clinical significance of tumor-infiltrating T cells based on PD-1 expression, their spatial organization, and the balance between subsets in a series of UCB patients. MethodsFlow cytometry for PD-1, CD4, and CD8 was performed in 6 UBC patients and 5 healthy donors. A series of 155 UBC patients with tissue slides were stained for triple color immunofluorescence. Stromal and intratumoral regions of the cancer tissue were respectively evaluated. Features derived from triple staining were analyzed for their correlations with clinical characteristics and patient prognosis. ResultsFlow cytometric analysis showed PD-1+ T cells were more frequently accumulated at the tumor site than in blood (p < 0.001). The proportion of PD-1+ T cells within CD4+ and/or CD8+ T cells is higher in the intratumoral region, as compared with the stroma by immunofluorescence evaluation (all p < 0.001, n = 155). Moreover, a high proportion of PD-1+ T cells within T cells in the intratumoral region, but not in the stroma, was predictive of a poorer overall survival (p = 0.0075) and recurrence-free survival (p = 0.0062), and was positively associated with aggressive clinical features (all p < 0.05). However, a low CD4/CD8 ratio among the PD-1+ T cells in the tumor stroma, but not in the intratumoral region, was significantly associated with shorter overall survival (p = 0.0164) and recurrence-free survival (p = 0.0016), which emerged as an independent predictor in multivariate analysis for UCB patients. ConclusionsTaken together, our results emphasize that PD-1 expression in T cell subsets, based on their topographic micro-localizations, provides valuable prognostic information for UCB patients.

Different orders of scattering through time-resolved Mueller matrix imaging estimates of pre-malignancy in human cervical tissues.

Time-resolved Mueller matrix (MM) imaging polarimetry in transmission mode has been implemented in both epithelium and stromal regions of cervical tissues to explore the various polarization dynamics in connection with the diagnosis of cervical precancer. The picosecond-resolved intensity patterns of various MM elements, resulting from the various orders of scattering, at different time delays provide clear demarcation between the epithelium and stroma of cervical tissue. The time dependent depolarization and retardance maps are seen to differentiate the epithelium from stroma. The average values of time dependent linear, linear-45, and circular depolarization and linear, circular, and scalar retardance parameters in different regimes of scattering from the optically anisotropic stromal region identify the pre-malignancy in cervical tissue. As the disease evolves, time dependent linear depolarization varies to larger values as compared to time dependent circular depolarization. Interestingly, the chirality of the collagen network that rotates the plane of polarized light in either direction in normal samples is limited to only the clockwise direction during the progression of the disease. These results show potential in the early detection and understanding of the mechanisms of morphological changes in cervical cancer development.

Spatial autocorrelation analysis on two-dimensional images of Mueller matrix for diagnosis and differentiation of cervical precancer.

The spatial autocorrelation and correlation map of amplitude and phase anisotropy along with depolarization parameter from the stroma of uterine cervix utilizing their Mueller matrix (MM) images have been reported for early diagnosis of cervical cancer and differentiation of precancerous stages. The comparative results of the evaluation of the spatial autocorrelation over MM images of optically anisotropic collagen structures from normal and various grades of cervical precancer reflect significant alterations which are correlated with the pathological changes. The spatially varying polarizance from different region of anisotropic stromal region gets correlated within a given spatial lag during the precancerous changes. The diattenuation governing elements M12, M13 and M14 clearly discriminate normal and various grades of precancerous cervical tissue through their autocorrelation profile and correlation map. Evaluation of autocorrelation of spatially varying linear birefringence and linear-45 birefringence characterized by MM elements M34 and M43 and M24 and M42 are not found to differ between the precancer grades, indicating that these changes may be arising from highly directional collagen network while the changes displayed by MM elements M23 and M32 faithfully represent that the chirality of the stromal region is compromised as the cervical cancer evolves and only one type of nature dominates.

IL-10 suppresses IFN-γ-mediated signaling in lung adenocarcinoma

Interleukin-10 (IL-10) is a pleiotropic cytokine produced by a wide variety of cells. It has been implicated in cancer progression, and at times, it has seemingly contradictory effects. The impact of IL-10 on immune components in the context of cancer has been intensively investigated, but its effect on cancer cells remains poorly understood. In this study, we examined the expression of IL-10 and IL-10 receptor 1 (IL-10R1) in resected locally advanced lung adenocarcinoma by immunohistochemistry. IL-10 immunoreactivity was stronger in intraepithelial regions than in stroma. The amount of IL-10 found either in intraepithelial or in stromal regions had no prognostic value, but the relative distribution of IL-10 in these two locations was related to cancer-immune phenotypes. High expression of IL-10R1 by tumor cells was significantly correlated with poor prognosis, suggesting that IL-10-mediated signaling may induce cancer cell intrinsic effects that promote cancer progression. Functional analysis using human lung adenocarcinoma cell lines revealed that IL-10 did not directly affect cell proliferation and migration. Incubation of cancer cells with IL-10 suppressed interferon-γ (IFN-γ)-induced STAT1 phosphorylation and inhibited the transcription of IFN-γ-targeted genes, such as CXCL9, CXCL10, and PD-L1. IL-10 enhanced IFN-γ-induced SOCS1 and SOCS3 expression, an effect that might be responsible for the downregulation of STAT1 activity in cancer cells. Our findings provide a rationale for targeting IL-10 on cancer cells as a potential strategy for treating cancer.

Abstract B65: Mathematical modeling studies on spatial profiles of tumor-infiltrating T cells

Abstract Activated cytotoxic T lymphocytes have been demonstrated to be able to kill antigen-specific cancer cells via various mechanisms. Not surprisingly, stronger infiltration of cytotoxic T cells into tumor/tumor-cell clusters generally associates with better prognosis, which has been demonstrated in various cancers. There have been efforts on quantifying the distribution of cytotoxic T cells on the whole tumor level. On the other hand, a solid tumor is usually composed of many tumor-cell clusters as well as stroma in gaps between those clusters. It has been noticed that T cells can be mostly localized in the stromal regions of a solid tumor. Therefore, it is also important to quantify the spatial pattern of T cells on the tumor-cell cluster level and further investigate the mechanism underlying the observed limited infiltration. In our work, we quantified the spatial distribution of CD8+ T cells with respect to their distance to the boundary of individual tumor-cell clusters. Generally, we observed that: i) patients differ in infiltration at the cluster level; ii) for most samples, T cells mainly accumulate outside the tumor-cell clusters; iii) for some samples T cells can effectively infiltrate the tumor-cell clusters; and iv) for other samples, the T-cell infiltration profile is intermediate. This last possibility reveals a non-monotonic distribution of T cells, i.e., a drop of T-cell density at the boundary coupled with a second accumulation of T cells at the center of tumor-cell clusters. Based on the quantified CD8+ T-cell profiles on the tumor-cell cluster level, we constructed mathematical models to test two hypothesized contributors affecting the spatial distribution of CD8+ T cells: a physical motility barrier set up by the ECM fibers in the stroma and a biochemical inhibitor ultimately due to the cancer cells inside the tumor-cell clusters. Mathematical models that only include physical barrier effects can qualitatively capture some (but not all) spatial features of the T-cell profiles. However, there is one significant shortcoming: the physical barrier scenario predicts that the profiles observed should be transient and hence eventually T cells should infiltrate all tumors. This appears inconsistent with simple time-scale estimates. A biochemical model focusing on T-cell repulsion can give rise to the observed spatial profiles as steady-state solutions; the different patterns correspond to different properties of cancer cells in different patients. We therefore favor this modeling framework. Of course, a definitive test would require experiments that would enable us to study the infiltration of T cells in a time-dependent manner or alternatively perturb possible mechanisms with drugs to directly test their effects on the infiltration pattern of T cells. Citation Format: Tina Gruosso, Morag Park, Herbert Levine, Xuefei Li. Mathematical modeling studies on spatial profiles of tumor-infiltrating T cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B65.

Consortum between mast cell and prostatic lesions-An etiological factor or mere coexistence

Introduction: Prostate cancer exhibits a complex genetic and heterogeneous histological pattern reflecting its multifactorial phenotype. Mast cells mainly known for their role in allergy and anaphylaxis, now regarded as key factor cell in angiogenesis, tissue remodeling and repair, whereas their importance in tumor pathology as either cancer promoter or inhibitor is still debated. Materials and Methods: Study included 100 cases of prostatic lesions with 74 cases of benign prostatic lesions and 26 cases of adenocarcinoma prostate, received in the form of transurethral resection of prostate chips for histopathological examination. The tissues were routinely processed, the paraffin sections stained by hematoxylin and eosin and 1% toluidine blue. Histological diagnosis was made. Mast cell count was done taking a standard of 10 high power fields(x40) and mean were calculated. Distributions of mast cells were noted in stroma, perivascular, periglandular and intraglandular regions. Results: Mast cell count was significantly higher in benign prostatic hyperplasia with mean of 3.6 per high power field whereas absence or low count was found in adenocarcinoma prostate with mean of 1.7 per high power field. Mast cells were located predominantly in stromal region in both BPH and adenocarcinoma prostate; in rest of the areas the numbers were comparable. Conclusion: In this study we observed significant increase in mast cell count in stroma of BPH compared to adenocarcinoma indicating that mast cell density is inversely proportional to malignancy. This suggests high mast cell count favors the prognosis. Keywords: Mast cells, Benign Prostatic Hyperplasia, Adenocarcinoma.

Age-related and zonal anatomical microstructure features of normal prostatic tissues: a preliminary study with VERDICT MRI.

This study aimed to explore the differences in VERDICT parameters for normal prostatic tissues among different regions and age groups. In total, 31 male volunteers were included in the study. Vascular, extracellular and restricted diffusion for cytometry in tumors (VERDICT) model parameters (fIC, intracellular volume fraction; fEES, extracellular-extravascular space fraction; and fVASC, vascular fraction) were calculated based on the whole prostate volume and the peripheral zone (PZ), central gland (CG) and anterior fibromuscular stroma(AS) regions of interest (ROIs). Agreement between two readers was determined by calculating the intraclass correlation coefficient. Two-way ANOVA was used to identify significant differences among age groups and regions within the prostate. Age-related and area-related changes in these parameters were assessed using Spearman's correlation coefficients. There were differences in the parameters among regions of the prostate (P < 0.05). In the PZ, there was a negative correlation between age and fIC (r = - 0.66, P < 0.001, adjusted P < 0.001), and age was positively correlated with fEES (r = 0.69, P < 0.001, adjusted P < 0.001) but had no correlation with fVASC. In the CG ROI, there was a positive correlation between age and fEES (r = 0.56, P < 0.001, adjusted P < 0.001) but no correlation between age and fVASC, similar to the results for the PZ. There were no correlations between age and VERDICT parameters in the AS. VERDICT parameters vary among different regions of the prostate and correlate with age. These findings are in agreement with histological theory.

Abstract P4-05-05: A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions

Abstract Background: The widespread adoption of breast cancer screening has increased the detection of pre-malignant lesions such as ductal carcinoma in situ (DCIS) but has not translated into increased survival rates. There are currently no reliable biomarkers to stratify DCIS into those which would not develop into an aggressive invasive cancer from those that do, and avoid over-treatment. The interaction with the DCIS microenvironment has likely been under-appreciated or not studied in the adequate clinical context. Importantly, the identification of novel biomarkers in DCIS is notoriously difficult. The rare and late recurrence to subsequent breast cancer (10% at 10 years) poses challenges in experimental design that only a few established, retrospective studies can solve. Most tissue specimens in the associated registries are small, fixed and archived in paraffin (FFPE), precluding their characterization via standard genomic assays. Here, we present an approach combining laser capture microdissection (LCM) with state-of-the-art genomic library preparation methods to enable molecular profiling from minute amounts of damaged nucleic acids and demonstrate its validity for the spatial molecular characterization of DCIS specimen. Methods: Targeted DNA sequencing (whole exome and cancer gene panel) was performed on limited dilutions (200, 50, 10,3 ng) of a test DNA from archived breast tissue, or validation DNA from 10 LCM regions from 3 DCIS specimens. Three library preparation strategies were evaluated, included adaptation of classic A-tailing (AT), or alternate single-strand (SS) or blunt-end (BE) adapter ligations. The expression profiling was performed using SMART-3SEQ protocol on 15 LCM regions from 8 DCIS specimen. Results: The use of an FFPE-optimized hybrid-capture AT protocol resulted in poor coverage performance (7% of targeted base pairs covered at ≥20x) from test DNA inputs lower than 50 ng and suggested the ligation of the sequencing adapter as the initial bottleneck. Alternatively steps using SS or BE adapter ligation from 3 ng of test DNA (~500 cells) increased the library complexity, reducing the fraction of duplicate reads and increasing the fraction of targeted base pairs covered at 20x to 49% and 100% respectively. Artifactual cytosine to tyrosine substitutions, a signature of formalin damage, were restricted to variants of low allelic fraction (&amp;lt;5%). As a result, the use of molecular barcodes, ensemble variant calling and heuristic variant filtering, resulted in 98% precision and 96% recall in variant calling accuracy from 3ng input, when compared to 200 ng input from a fresh frozen mirrored specimen. Similarly, the presence of an ERBB2 amplification was validated in all tested conditions. The validation of the BE approach on 10 LCM regions from 3 DCIS showed decreasing levels of clonal relationships when comparing regions from adjacent sections, then from matching distinct regions, then from distinct specimen. The heterogeneity of TP53 loss of heterozygosity within a specimen was consistent with high-grade. Furthermore, gene expression profiling of 15 regions from 8 archived DCIS specimen differentiated expression profiles of stromal and epithelial regions, within and between specimen, highlighting sets of genes associated with DCIS subtype and the immune make-up of the micro-environment. Conclusion: The proposed approach - from coordinated specimen preparation to mutational and expression profiling - was therefore validated to support the spatial characterization of pre-cancerous lesions and the microenvironment in which they evolve. Compatible with a limited quantity of tissue from archived specimens, the approach sets a new paradigm for the retrospective molecular characterization of specimen with longitudinal follow-up and associated long-term outcome. Citation Format: Daniela Nachamanson, Joseph Steward, Taeyong Kim, Huazhen Yao, Eliza Jeong, Thomas O'Keefe, Gillian L. Hirst, Farnaz Hasteh, Kristen Jepsen, Laura J. Esserman, Alexander Borowsky, Olivier Harismendy. A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-05.

Mapping of retardance, diattenuation and polarizance vector on Poincare sphere for diagnosis and classification of cervical precancer.

The mapping of diattenuation, polarizance and retardance vector (normalized Stokes vector) on Poincare sphere, evaluated from Mueller matrix of optically anisotropic stromal region of cervical tissues, is presented for cervical precancer detection and its staging. This reveals that the changes in the polarization states shown by these normalized Stokes vectors corresponds to the degradation of linearly arranged collagen fibers, breakage of the collagen cross links in the stromal region and change in the density of scattering sites when cervical cancer evolves. The distinct nature of real and imaginary parts of the refractive index for linear, linear-45 and circular polarization from the optically anisotropic stromal region underscore the various polarization structures of the connective tissue region which get modified during the pathological changes. It has been found that versatility of these vectors for normal and precancerous cervical tissue of various grades may be utilized as a key distinction for qualitative staging of cervical precancer tissue. Quantitative classification of precancerous stages of cervical precancer has been determined with 95%-100% sensitivity and 93%-100% specificity through the evaluation of linear and circular diattenuation, linear polarizance and linear birefringence from the components of the respective vectors.

Assessment of anisotropy of collagen structures through spatial frequencies of Mueller matrix images for cervical pre-cancer detection.

Analysis of spatial frequency of Mueller matrix (MM) images in the Fourier domain yields quantifying parameters of anisotropy in the stromal region in normal and precancerous tissue sections of human uterine cervix. The spatial frequencies of MM elements reveal reliable information of microscopic structural organization arising from the different orientations of collagen fibers in the connective tissue and their randomization with disease progression. Specifically, the local disorder generated in the normal periodic and regular structure of collagen during the growth of the cervical cancer finds characteristic manifestation in the Fourier spectrum of the selected Mueller matrix elements encoding the anisotropy effects through retardance and birefringence. In contrast, Fourier spectra of differential polarization gated images are limited to only one orientation of collagen. Fourier spectra of first row elements M11, M12, M13, and M14 and first column elements M11, M21, M31, and M41 discriminates cervical inter-epithelial neoplasia (CIN)-I from normal cervical tissue samples with 95%-100% sensitivity and specificity. FFT spectra of first and fourth row elements classify CIN-I and CIN-II grades of cervical cancerous tissues with 90%-100% sensitivity and 87%-100% specificity. Normal and CIN-II grade samples are successfully discriminated through Fourier spectra of every MM element while that of M31 element arises as the key classifier among normal, CIN-I, and CIN-II grades of cervical cancer with 100% sensitivity and specificity. These results demonstrate the promise of spatial frequency analysis of Mueller matrix images as a novel, to the best of our knowledge, approach for cancer/precancer detection.

IL-6 and G-CSF production resulting from lung cancer in an HIV patient.

Increasing reports have noted an increased prevalence of lung cancer in human immunodeficiency virus (HIV)-positive patients with poor prognosis. A 51-year-old HIV-positive man was diagnosed with stage IV squamous cell lung cancer. He had high grade spike intermittent fever and persistent elevation of the white blood cell count as well as C-reactive protein (CRP) levels. Although we suspected opportunistic infections, we did not detect any infection. The autopsy showed positive immunostaining for Interleukin-6 (IL-6) in plasma cells of the stromal regions and G-CSF in tumor cells, which were considered responsible for his significant tumor fever, leukocytosis and high titers of CRP. This case report highlights the need to consider cytokine-producing tumor as a differential diagnosis of fever and high inflammatory status in HIV-positive cancer patients.