Stromal Density Research Articles

The Prognostic Role of Macrophage Polarization in the Colorectal Cancer Microenvironment.

Macrophages are among the most common cells in the colorectal cancer microenvironment, but their prognostic significance is incompletely understood. Using multiplexed immunofluorescence for CD68, CD86, IRF5, MAF, MRC1 (CD206), and KRT (cytokeratins) combined with digital image analysis and machine learning, we assessed the polarization spectrum of tumor-associated macrophages in 931 colorectal carcinomas. We then applied Cox proportional hazards regression to assess prognostic survival associations of intraepithelial and stromal densities of M1-like and M2-like macrophages while controlling for potential confounders, including stage and microsatellite instability status. We found that high tumor stromal density of M2-like macrophages was associated with worse cancer-specific survival, whereas tumor stromal density of M1-like macrophages was not significantly associated with better cancer-specific survival. High M1:M2 density ratio in tumor stroma was associated with better cancer-specific survival. Overall macrophage densities in tumor intraepithelial or stromal regions were not prognostic. These findings suggested that macrophage polarization state, rather than their overall density, was associated with cancer-specific survival, with M1- and M2-like macrophage phenotypes exhibiting distinct prognostic roles. These results highlight the utility of a multimarker strategy to assess the macrophage polarization at single-cell resolution within the tumor microenvironment.

IRIS CHANGES AT PATIENTS WITH TEMPORОMANDIBULAR JOINT DISEASES AND URINARY SYSTEM PATHOLOGY

Relevance. In recent years, many have been devoted to the problem of the temporomandibular joint (TMJ) diseases, in which the attention is paid to the widespread pathology of TMJ in young people, which develops against the background of the genetically-determined weakness of connective tissue (CTs), which is also the present occurrence of various concomitant diseases of polygenic-multifactorial nature, including the organs of the urinary system (US).
 The study of the state of the iris is used as a screening technique that allows you to quickly, simply, harmlessly, informatively, painlessly, non-invasively diagnose the genetically determined structural and functional state and quality of the CTs.
 Objective. To investigate the iris changes in patients with TMJ and US diseases, to reveal the dependence of the occurrence of degenerative-dystrophic and destructive-inflammatory changes in the joints and US on the structural and functional state of the CTs, to supplement the known traditional methods of diagnosing genetically determined CTs weakness.
 Materials and methods. The study involved 54 patients (men – 14, women – 40), whose average age was 37.3±7.6 years, who were treated at the Dental Medical Center of the Bogomolets NMU. Control group – 22 patients (men – 10, women – 12) without general somatic pathology, with a physiological bite, without signs of TMJ diseases, whose average age was 25.7±6.8 years. Main group – 22 patients (men – 4, women – 28) with TMJ diseases and US pathology, whose average age was 31.6±7.7 years.
 Iridobiomicroscopy was performed in patients of both groups. Iridogenetic constitutional signs were determined: the color of eyes, constitutional type after E.S. Velkhover, type with the connective tissue weakness after I. Deck, the density of iris stoma.
 The obtained laboratory data were referenced in the International System of Units and processed by variational statistics using MedStat and EZR v.1.35 (Saitama Medical Center, Jichi Medical University, Saitama, Japan, 2017), which is a graphical interface to RFSC (The R Foundation for Statistical Computing, Vienna, Austria).
 Results. Among the examined patients with TMJ diseases and MVS pathology, 75% showed predominantly light eye color and V degree (46.9%) of iris stroma density, in the control group: dark iris color (54.5%) and II degree (68.2%) of iris density.
 Iridogenetic constitutional signs of the CT weakness in the patients with TMJ diseases have been determined: the light color of eyes, radial-lacunar constitutional type after E.S. Velkhover (75%), lymphatic constitutional type with the connective tissue weakness after I. Deck (57,9%).
 Structural local or chromatic changes of the iris stroma in the projection region of the kidney (75%) and bladder (43.6%) in young patients with TMJ diseases showed a congenital weakness of the CTs of these organs and a tendency to develop pathology of the US.
 Conclusions. Patients with degenerative-dystrophic and destructive-inflammatory diseases of the TMJ and concomitant US pathology are characterized by iridogenetic constitutional signs of the CTs weakness in the patients with TMJ diseases have been determined: the light color of eyes, radial-lacunar constitutional type after E.S. Velkhover, low degree of iris stroma density, lymphatic constitutional type with the connective tissue weakness after I. Deck.
 Structural local or chromatic changes of the iris stroma in the projection region of the kidney and bladder in young patients with TMJ diseases showed a congenital weakness of the CTs of these organs and a tendency to develop pathology of the US.
 It can be assumed that the development of TMJ diseases in young patients is based on dysplastic changes in the CTs system, which is additionally manifested in the examined patients by pathological changes in the US.
 Iridobiomicroscopy, as a screening technique for determining the structural and functional state of CTs, makes it possible to increase the accuracy of diagnostics when examining patients with TMJ diseases who have concomitant pathology of internal organs, including US.

Primary tumors from mucosal barrier organs drive unique eosinophil infiltration patterns and clinical associations

ABSTRACT Eosinophils are bone marrow-derived granulocytes that display key effector functions in allergic diseases. Nonetheless, recent data highlight important roles for eosinophils in the tumor microenvironment (TME). Eosinophils have been attributed with pleiotropic and perhaps conflicting functions, which may be attributed at least in part to variations in eosinophil quantitation in the TME. Thus, a reliable, quantitative, and robust method for the assessment of eosinophilic infiltration in the TME is required. This type of methodology could standardize the identification of these cells and promote the subsequent generation of hypothesis-driven mechanistic studies. To this end, we conducted a comprehensive analysis of multiple primary tumors from distinct anatomical sites using a standardized method. Bioinformatics analysis of 10,469 genomically profiled primary tumors revealed that eosinophil abundance within different tumors can be categorized into three groups representing tumors with high, intermediate, and low eosinophil levels. Consequently, eosinophil abundance, as well as spatial distribution, was determined in tissue tumor arrays of six tumors representing all three classifications (colon and esophagus – high; lung – intermediate; cervix, ovary, and breast – low). With the exception of breast cancer, eosinophils were mainly localized in the tumor stroma. Importantly, the tumor anatomical site was identified as the primary predictive factor of eosinophil stromal density highlighting a distinction between mucosal-barrier organs versus non-mucosal barrier organs. These findings enhance our understanding of eosinophil diversity in the TME and provide a compelling rationale for future experiments assessing the activity of these cells.

The scattered puzzle effect: implantation disorders in chronic endometritis

Chronic endometritis (CE) is defined as a state of inflammation localized in the endometrium, accompanied by edema, dissociated maturation of epithelial cells and fibroblasts, increased stromal density and the presence of plasma cell infiltrate in it. The connection between chronic inflammation in the endometrium and infertility deserves special attention. Inadequate response of immunocompetent endometrial cells, including impaired synthesis of proinflammatory cytokines, dysreceptiveness, disorders of proliferation and differentiation processes are the main links in the formation of infertility in patients with CE. Despite the fact that the presence of a normocenosis of the uterine cavity today is not in doubt this is a physiological norm, persistent bacterial infection of the endometrium is still called the main etiopathogenetic factor of CE and, therefore, the main point of application of therapeutic agents. Nevertheless, a number of works have emphasized the special role of not bacterial, but viral etiology of endometritis, especially in the context of infertility developing against this background. It seems that the role of viral endometrial infection in adverse pregnancy outcomes and in vitro fertilization programs is underestimated. Further research is needed to clarify the relationship of viral infection as a trigger of implantation failure in infertile women with CE.

The Prognostic Value of Stromal Tumor-Infiltrating Lymphocytes in Intrahepatic Cholangiocarcinoma

Objective: We conducted this study to assess the predictive value of tumor-infiltrating lymphocytes (TILs) for progression-free survival (PFS) and overall survival (OS) in intrahepatic cholangiocarcinoma (ICC) patients after complete resection. Methods: Sixty-eight patients with resectable ICC were included in this study. We studied stromal TIL density and scored it by staining sections from surgically resected ICC patients with hematoxylin and eosin (HE). The clinical data and prognosis of ICC patients were obtained by searching clinical and follow-up records. Results: A stromal TIL negative status was an independent predictor of poor OS (HR=2.84, 95% CI 1.48- 5.46, P<0.01) and poor PFS (HR=2.88, 95% CI 1.44-5.76, P<0.01). Low stromal TIL density was associated with high CA125 (P=0.03) and CA19-9 (P<0.01) levels. Patients with a stromal TIL negative status tended to develop tumors with a high level of CA19-9 (P=0.05), high differentiation (P=0.02), a large diameter (P=0.05), a positive bile duct/vascular cancer embolus (P=0.03) and positive satellite nodules (P=0.02). Conclusion: Our data suggest that stromal TILs play an important role in predicting the PFS and OS of ICC patients after complete resection.

Abstract PO-030: Deep-learning image-based tumor, stroma, and lymphocytes spatial relationships and clinicopathological features that affect survival in pancreatic cancer patients

Abstract Background: Stromal and lymphocyte density have each been implicated in differential survival in pancreatic cancer (Torphy, 2018 & Orhan, 2020). In this study, we developed an automated deep-learning system to provide risk-assessment upon spatial relationships between tumor, stroma, and lymphocyte regions in pancreatic pathology images. Methods: Diagnostic H&E-stained pathology images from 82 pancreatic adenocarcinoma patients who underwent chemotherapy were acquired from TCGA sources. Thirty-two patients were held out for testing purposes. Tumor, stroma, and lymphocytes image masks were generated using pre-trained convolutional neural networks, and their co-distribution was summarized in nine numerical image-based features. Optimal thresholds in these image-based features were identified using 2-way Gaussian mixture models. This process found four spatial image features that significantly contributed to low-risk of early death: Low lymphocyte count, lymphocytes adjacent to tumor regions, and stromal adjacency to tumor regions. Ability to separate patients based on these features was evaluated using silhouette score, concordance index, and Cox proportional hazards ratios (HR). Results: Without using image-based features, exhaustive search of this cohort’s clinicopathological features found an optimal Cox proportional hazards model can yield a HR = 0.22 (p = 0.02) in 50 training examples and HR = 0.41 (p = 0.29) on 32 unseen test patients, ultimately utilizing just pathologically-determined T, and N information. The developed image-based risk predictor improved performance with HR = 0.51 (p = 0.06) on training data and HR = 0.52 (p = 0.09) on unseen test data. Combining the image-based risk models to selected clinicopathological features enhanced performance further to HR = 0.25 (p = 0.01) on the training set and HR = 0.37 (p = 0.07) on unseen test patients. Conclusions: Our interpretable image-based risk predictor shows high-risk pancreatic cancer patients have higher lymphocyte count overall but proportionally fewer tumor-infiltrating lymphocytes (TILs). In addition, this system shows high-risk patients have less stromal tissue within 100um from tumor compared to low risk patients. By aggregating both standard clinicopathological features with the proposed image-based risk assessment, superior separation in survival curves was achieved for both training and testing sets compared to either risk-model alone. Thus, our study demonstrates that image-based risk-associated features are independently prognostic of clinicopathological features. Despite the very limited sample-size of similarly-treated patients within the training dataset, these results trend towards significance and warrant further study within a larger cohort. Citation Format: Mustafa I. Jaber, Liudmila Beziaeva, Robert J. Torphy, Stephen C. Benz, Shahrooz Rabizadeh, Patrick Soon-Shiong, Christopher W Szeto. Deep-learning image-based tumor, stroma, and lymphocytes spatial relationships and clinicopathological features that affect survival in pancreatic cancer patients [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-030.

Alignment of stromal ECM fibers and microvessel density determine overall survival in pancreatic cancer - an analysis of stroma morphology –

Alignment of stromal ECM fibers and microvessel density determine overall survival in pancreatic cancer - an analysis of stroma morphology –

High density of stroma-localized CD11c-positive macrophages is associated with longer overall survival in high-grade serous ovarian cancer

ObjectivePre-clinical studies have identified marker- and tumor compartment-defined functionally distinct macrophage subsets. Our study analyzes marker-defined macrophage subsets in different tumor compartments of high-grade serous ovarian cancer (HGSC). MethodsA discovery cohort (N = 113) was subjected to immunohistochemistry (IHC) analyses. CD68-positivity was confirmed for CD11c-, CD80- and CD163-positive cells. Subset-marker-positive cells were scored in the total tumor and in four tumor compartments. Correlation analyses investigated co-expression of subsets, relationship to CD8+ cells and survival associations. A validation cohort (N = 121) was used to confirm selected findings from the discovery cohort. ResultsCD163-positve cells was the most abundant subtype in all compartments. CD11c and CD163 subsets were strongly correlated with each other in stroma and epithelial areas, whereas CD80 and CD163 were correlated in epithelial areas. CD80 and CD11c in perivascular areas showed low correlations. Strong associations were detected between CD8 and CD80 in the tumor epithelium-dominated areas, and between CD8 and CD11c in stroma areas. High stromal CD11c density was associated with a longer median overall survival in the discovery cohort (HR 0.39; CI 95%, 0.23–0.68; p = 0.001) and in the validation cohort (HR 0.46; CI 95%, 0.22–0.93; p = 0.03). ConclusionsOur study supports the existence of clinically relevant marker- and localization defined macrophage subsets in HGSC, which are independently regulated. Moreover, it suggests stromal CD11c as a novel prognostic marker in HGSC.

Intratumoral CD103-positive tumour-infiltrating lymphocytes are associated with favourable prognosis in patients with triple-negative breast cancer.

Cluster of differentiation 103 (CD103), a marker of tissue resident memory T cells, is expressed on subsets of CD8+ T lymphocytes. We investigated the prognostic significance of CD103+ intra-epithelial tumour-infiltrating lymphocytes (iTILs) in invasive breast cancer (IBC). Immunohistochemistry was performed for CD103, CD8 and TGF-β isoforms (1, 2 and 3) on tissue microarrays of 1187 IBC samples. CD103+ and CD8+ iTILs were present in 904 (76.2%) and 854 (74%) cases with an overall mean±standard deviation of 38.2±100.2/mm2 and 30.4±89.7/mm2 , respectively. The numbers of CD103+ and CD8+ iTILs were positively correlated, and CD103+ iTILs outnumbered CD8+ iTILs in HER2-positive and triple-negative breast cancer (TNBC). CD103+ and CD8+ iTIL densities were significantly higher in tumours of histological grade 3, absence of lymphovascular invasion, high Ki-67 index, high stromal TIL density or TGF-β3 expression. High CD103+ iTIL density was associated with better disease-free survival (DFS, P=0.007), but no significant association was observed for overall survival (OS). Subgroup analysis by cancer molecular subtype showed that CD103+ iTIL count was prognostic only for TNBC (OS, P=0.035; DFS, P=0.009). CD8+ iTIL density was significant for DFS, but not for OS, in the entire cohort and TNBC. In multivariate analysis, CD103+ iTIL density was an independent prognostic factor of OS (P=0.02) and DFS (P=0.007) in TNBC, while CD8+ iTIL density was not significant for survival. CD103 iTIL density can serve as a predictor of good prognosis in patients with TNBC.

Coffee Intake and Colorectal Cancer Incidence According to T-Cell Response.

We hypothesized that the associations between coffee intake and colorectal cancer (CRC) incidence might differ by immune cell densities in CRC tissue. Using the Nurses’ Health Study and the Health Professionals Follow-up Study, we examined the association of coffee intake with incidence of CRC classified by intraepithelial or stromal T-cell subset densities by multiplex immunofluorescence assay for CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3. We applied an inverse probability-weighted Cox proportional hazardsregression model to control for selection bias and potential confounders. During follow-up of 133 924 participants (3 585 019 person-years), we documented 3161 incident CRC cases, including 908 CRC cases with available data on T-cell densities in tumor tissue. The association between coffee intake and CRC was not statistically significantly different by intraepithelial or stroma T-cell subset (Pheterogeneity > .38). Hence, there is no sufficient evidence for differential effect of coffee intake on incidence of CRC subtypes classified by T-cell infiltrates.

Prognostic Significance of Immune Cell Populations Identified by Machine Learning in Colorectal Cancer Using Routine Hematoxylin and Eosin-Stained Sections.

Although high T-cell density is a well-established favorable prognostic factor in colorectal cancer, the prognostic significance of tumor-associated plasma cells, neutrophils, and eosinophils is less well-defined. We computationally processed digital images of hematoxylin and eosin (H&E)-stained sections to identify lymphocytes, plasma cells, neutrophils, and eosinophils in tumor intraepithelial and stromal areas of 934 colorectal cancers in two prospective cohort studies. Multivariable Cox proportional hazards regression was used to compute mortality HR according to cell density quartiles. The spatial patterns of immune cell infiltration were studied using the GTumor:Immune cell function, which estimates the likelihood of any tumor cell in a sample having at least one neighboring immune cell of the specified type within a certain radius. Validation studies were performed on an independent cohort of 570 colorectal cancers. Immune cell densities measured by the automated classifier demonstrated high correlation with densities both from manual counts and those obtained from an independently trained automated classifier (Spearman's ρ 0.71-0.96). High densities of stromal lymphocytes and eosinophils were associated with better cancer-specific survival [P trend < 0.001; multivariable HR (4th vs 1st quartile of eosinophils), 0.49; 95% confidence interval, 0.34-0.71]. High GTumor:Lymphocyte area under the curve (AUC0,20μm; P trend = 0.002) and high GTumor:Eosinophil AUC0,20μm (P trend < 0.001) also showed associations with better cancer-specific survival. High stromal eosinophil density was also associated with better cancer-specific survival in the validation cohort (P trend < 0.001). These findings highlight the potential for machine learning assessment of H&E-stained sections to provide robust, quantitative tumor-immune biomarkers for precision medicine.

Abstract CT118: Phase I study of defactinib combined with pembrolizumab and gemcitabine in patients with advanced cancer: Experiences of pancreatic ductal adenocarcinoma (PDAC) patients

Abstract Background: Focal adhesion kinase (FAK) is consistently hyperactivated in multiple tumor types including PDAC. Our preclinical work showed that FAK and PD-1 inhibitors elicit significant tumor regression, and that combining FAK and PD-1 inhibitors with gemcitabine achieves a maximal response. This suggests the need for a cytotoxic agent to bolster antigen presentation. Defactinib is an orally available, well-tolerated, potent FAK inhibitor. Methods: This study included a dose escalation phase (all solid tumors) and dose expansion phase (metastatic PDAC). A 3+3 design with five dose levels was used in the dose escalation phase. For the expansion phase, 10 PDAC patients with stable disease on front-line gemcitabine/nab-paclitaxel were enrolled in the Maintenance cohort (A), while 10 patients who progressed on at least one line of therapy were enrolled in the Refractory cohort (B). Pre and on-treatment biopsied were collected from all PDAC patients. The primary endpoint was to determine the recommended phase 2 dose (RP2D). Secondary endpoints included safety, toxicity, objective response rate, progression-free survival (PFS) and overall survival (OS). The exploratory endpoints included developing a molecular and immune profile for treatment response. Results: In dose escalation, the common low grade (G1/2) treatment-emergent adverse events included fatigue, anorexia, nausea and vomiting. No DLTs were seen. The RP2D was defactinib 400mg twice daily, gemcitabine 1000 mg/m2 days 1, 8 and pembrolizumab 200mg day 1 of a 21-day cycle. Safety and toxicity results in the escalation cohort were reported at the 2018 ASCO Annual Meeting. Among 8 evaluable PDAC patients enrolled on the dose escalation phase, we observed 1 (13%) partial response (PR), 3 (38%) stable disease (SD), 4 (50%) progressive disease (PD). In the Maintenance cohort, 1 (10%) PR, 6 (60%) SD, and 3 (30%) PD were observed. Two patients in the Maintenance cohort remain on study, and the median time on treatment was 4.6 months. In the Refractory cohort, 5 (50%) had SD, 4 (40%) PD, 1 not evaluable; median PFS was 2.9 months and median OS was 7.6 months. Interestingly, two confirmed PR patients both have MSS disease. Paired biopsies in PDAC patients show increased proliferating CD8+ T cells, while Tregs, macrophages, and stromal density decrease with treatment. Conclusions: The combination regimen was well tolerated. Encouraging efficacy signals were observed in both Maintenance and Refractory cohorts. Updated outcomes and correlative analyses are forthcoming. Citation Format: Andrea Wang-Gillam, Robert McWilliams, A. Craig Lockhart, Benjamin R Tan, Rama Suresh, Kian-Huat Lim, Katrina S. Pedersen, Nikolaos Trikalinos, Olivia Aranha, Haeseong Park, Lee Ratner, Nicholas Boice, David G. Denardo. Phase I study of defactinib combined with pembrolizumab and gemcitabine in patients with advanced cancer: Experiences of pancreatic ductal adenocarcinoma (PDAC) patients [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 CT118.

Corneal In Vivo Laser-Scanning Confocal Microscopy Findings in Dry Eye Patients with Sjögren's Syndrome.

Purpose: To evaluate the changes in cornea in Sjögren’s syndrome (SS) with a novel confocal microscopy device. Methods: Twenty-three right eyes of patients with SS (23 women; mean age, 65.4 ± 11.4 years) and 13 right eyes of 13 age- and sex-matched control subjects (13 women; mean age, 68.8 ± 9.8 years) were studied. Furthermore, eight right eyes of patients with SS (8 women; mean age, 66.9 ± 9.6 years) were studied to evaluate the corneal microscopic alterations after the treatment with topical 3% diquafosol sodium eye drops. All cases had tear quantity, tear breakup time (BUT), ocular surface staining measurements, and corneal in vivo laser-scanning confocal microscopy examinations. The density and area of corneal epithelial cells (superficial, wing, and basal), density of corneal stromal cells (anterior, intermediate, and posterior), density and area of corneal endothelial cells, density and morphology of corneal sub-basal nerve plexus, density of corneal sub-basal inflammatory cells were also assessed. Results: The tear quantity, stability, and vital staining scores were significantly worse in patients with SS than in control subjects (p < 0.0001). Corneal superficial epithelial cell density was significantly lower in SS compared with control subjects (p < 0.0001). Corneal superficial epithelial cell area was significantly larger in SS compared with control subjects (p = 0.007). Corneal sub-basal nerve fiber density was lower in SS compared with control subjects (p < 0.0001). Morphological abnormality of nerve fibers was observed in SS patients. Corneal sub-basal inflammatory cell density was significantly higher in SS patients compared with control subjects (p < 0.0001). Furthermore, the mean corneal superficial epithelial cell density and area, inflammatory cell density, corneal sub-basal nerve fiber density, and morphological abnormality of nerve fibers, were improved with topical 3% diquafosol sodium treatment in the dry eye patients with SS (p < 0.05). Conclusions: The diagnostic modality using in vivo laser-scanning confocal microscopy was a useful method for the evaluation of the corneal cell density and area, nerve fiber density and morphology, and inflammatory cell density in patients with SS and also a useful tool in the assessment of treatment effect with topical 3% diquafosol sodium in the SS patients.

Significance of SOX18 expression in nonmelanoma skin cancers for prediction of high-risk patients: a preliminary study.

SOX18 is an integral transcription factor that is involved in endothelial cells differentiation during both angiogenesis and lymphangiogenesis. Therefore, it has been implicated in tumor progression and metastasis. To study SOX18 expression in nonmelanoma skin cancers (NMSCs) in comparison to seborrheic keratosis (SK) and normal control skin, and to assess its probable role in tumor evolution and progression. This study was conducted on 60 specimens of NMSCs: 30 basal cell carcinomas (BCC) and 30 squamous cell carcinomas (SCC), 30 specimens of SK, and 30 normal skin specimens. All were examined for immunohistochemical expression of SOX18 antibody. Additionally, morphometric assessment of vessel density (blood & lymphatic) in each specimen was estimated. Significant SOX18 overexpression was observed in all studied cutaneous tumors in comparison to control skin. The highest score of SOX18 expression was detected in SCC, then BCC, and the least expression was reported in SK with significant difference between them. Furthermore, significant upregulation of SOX18 expression was observed in high-risk types of both BCC and SCC compared to low-risk types. Stromal vessel density showed significant differences between the studied tumors with the highest mean value in SCC, followed by BCC and then SK. Positive correlation between SOX18 expression in the studied tumors and their vessel density was detected. SOX18 may have a potential role in the evolution as well as progression of NMSCs, possibly through induction of both angiogenesis and lymphangiogenesis. Furthermore, it could be beneficial for prediction of NMSC patients with poor prognosis.

Alignment of stroma fibers, microvessel density and immune cell populations determine overall survival in pancreatic cancer-An analysis of stromal morphology.

IntroductionThe aim of this study was to define histo-morphological stroma characteristics by analyzing stromal components, and to evaluate their impact on local and systemic tumor spread and overall survival in pancreatic ductal adenocarcinoma (PDAC).Methods and materialsPatients who underwent oncologic resections with curative intent for PDAC were identified from a prospectively maintained database. Histological specimens were re-evaluated for morphological stroma features as stromal fibers, fibroblast morphology, stroma matrix density, microvessel density and distribution of immune cell populations.ResultsA total of 108 patients were identified undergoing curative resection for PDAC in the period from 2011–2016. 33 (30.6%) patients showed parallel alignment of stroma fibers while 75 (69.4%) had randomly oriented stroma fibers. As compared to parallel alignment, random orientation of stroma fibers was associated with larger tumor size (median 3.62 cm vs. median 2.87cm, p = 0.037), nodal positive disease (76.0% vs. 54.5%, p = 0.040), higher margin positive resection rates (41.9% vs. 15.2%, p = 0.008) and a trend for higher rates of T3/4 tumors (33.3% vs. 15.2%, p = 0.064). In univariate analysis, patients with parallel alignment of stroma fibers had improved overall survival rates as compared to patients with random orientation of stroma fibers (42 months vs. 22 months, p = 0.046). The combination of random orientation of stroma fibers and low microvessel density was associated with impaired overall survival rates (16 months vs. 36 months, p = 0.019). A high CD4/CD3 ratio (16 months vs. 33 months, p = 0.040) and high stromal density of CD163 positive cells were associated with reduced overall survival (27 months vs. 34 months, p = 0.039). In multivariable analysis, the combination of random orientation of stroma fibers and low microvessel density (HR 1.592, 95%CI 1.098–2.733, p = 0.029), high CD4/CD3 ratio (HR 2.044, 95%CI 1.203–3.508, p = 0.028) and high density of CD163 positive cells (HR 1.596, 95%CI 1.367–1.968, p = 0.036) remained independent prognostic factors.ConclusionAlignment of stroma fibers and microvessel density are simple histomorphological features serving as surrogate markers of local tumor progression dissemination and surgical resectability and determine prognosis in PDAC patients. High CD4/CD3 ratio and CD163 positive cell counts determine poor prognosis.

The predictive potential of the spatial signature of lymphocytes in breast cancer patients.

530 Background: Tumor-infiltrating lymphocytes in breast cancer have emerged as both a prognostic and a potentially predictive immunotherapy biomarker. Advancements in artificial intelligence can extract pathology-based spatial immune fingerprints for use as treatment decision support tools. Methods: We examined 908 primary breast cancer patients with whole slide images (WSI) available from TCGA database. Digital structuring of WSIs included automated detection of lymphocytes, tumor and tumor adjacent stroma, using deep learning-based semantic segmentation. Prognosis was defined as progression free interval (PFI). A Cox Survival analysis was used to detect prognostic spatial features. We used principal component analysis (PCA) to reduce and decorrelate significant features. The resulting PCA features were used to fit the final model. The model was then validated on an independent database of WSI of breast lumpectomies, from two tertiary hospitals in Israel. Results: The analysis included 908 WSI. The average age was 58.4 years old, with a majority of early stage breast cancer (76.7%, stage I and II). The detection performance for tumor area and lymphocytes reached F1 scores of 99% and 97% respectively, in comparison to human annotation. In the Kaplan Meier (KM) analysis of 414 early stage luminal breast cancers, a high number of lymphocyte clusters (LC) and a high ratio between stromal lymphocyte density and tumor lymphocyte density (LD-S/LD-T) were significantly associated with longer PFI (p = 0.005 and p = 0.038, respectively). Based on these features, two continuous PCA features were added to the multivariate model, and remained significantly associated with PFI after adjusting for age (HR = 1.19, 95% CI 1.05-1.35; HR = 1.26 95% CI 1.03-1.55). The validation set was underpowered (n = 79) and data is still being collected. In a preliminary KM analysis of 37 early stage luminal breast cancer cases from the validation set, LD-S/LD-T was significantly associated with longer PFI (p = 0.046). Conclusions: In our study, LC and LD-S/LD-T, presumably surrogate measures of peritumoral lymphocytes, were found significantly associated with longer PFI.

Assessment of associations between clinical and immune microenvironmental factors and tumor mutation burden in resected nonsmall cell lung cancer by applying machine learning to whole-slide images.

BackgroundIt is unclear whether clinical factors and immune microenvironment (IME) factors are associated with tumor mutation burden (TMB) in patients with nonsmall cell lung cancer (NSCLC).Materials and methodsWe assessed TMB in surgical tumor specimens by performing whole exome sequencing. IME profiles, including PD‐L1 tumor proportion score (TPS), stromal CD8 tumor‐infiltrating lymphocyte (TIL) density, and stromal Foxp3 TIL density, were quantified by digital pathology using a machine learning algorithm. To detect factors associated with TMB, clinical data, and IME factors were assessed by means of a multiple regression model.ResultsWe analyzed tumors from 200 of the 246 surgically resected NSCLC patients between September 2014 and September 2015. Patient background: median age (range) 70 years (39‐87); male 37.5%; smoker 27.5%; pathological stage (p‐stage) I/II/III, 63.5/22.5/14.0%; histological type Ad/Sq, 77.0/23.0%; primary tumor location upper/lower, 58.5/41.5%; median PET SUV 7.5 (0.86‐29.8); median serum CEA (sCEA) level 3.4 ng/mL (0.5‐144.3); median serum CYFRA 21‐1 (sCYFRA) level 1.2 ng/mL (1.0‐38.0); median TMB 2.19/ Mb (0.12‐64.38); median PD‐L1 TPS 15.1% (0.09‐77.4); median stromal CD8 TIL density 582.1/mm2 (120.0‐4967.6);, and median stromal Foxp3 TIL density 183.7/mm2 (6.3‐544.0).The multiple regression analysis identified three factors associated with higher TMB: smoking status: smoker, increase PET SUV, and sCEA level: >5 ng/mL (P < .001, P < .001, and P = .006, respectively).ConclusionsThe IME factors assessed were not associated with TMB, but our findings showed that, in addition to smoking, PET SUV and sCEA levels may be independent predictors of TMB. TMB and IME factors are independent factors in resected NSCLC.

Prognostic impact of stromal and intratumoral CD3, CD8 and FOXP3 in adjuvantly treated breast cancer: do they add information over stromal tumor-infiltrating lymphocyte density?

Tumor-infiltrating lymphocytes (TILs) and their subsets contribute to breast cancer prognosis. We investigated the prognostic impact of CD3+, CD8+ and FOXP3+ TILs in patients with early intermediate/high-risk breast cancer treated with adjuvant anthracycline-based chemotherapy within two randomized trials conducted by our Group. We examined 1011 patients (median follow-up 130.9months) and their tumors for total, stromal (s) and intratumoral (i) CD3, CD8 and FOXP3 lymphocyte density (counts/mm2) on tissue-microarray cores by immunohistochemistry. Morphological sTIL density on whole H&E-stained sections was also evaluated. The majority of TILs were CD3+. Total CD3 and CD8, sCD3 and sCD8, iCD3 and iCD8, sFOXP3 and iFOXP3 were strongly correlated (Spearman's rho values > 0.6). High individual lymphocytic subsets and sTIL density were strongly associated with high tumor grade, higher proliferation and HER2-positive and triple-negative tumors (all p values < 0.001). Higher sTIL density (10% increments), high density of almost each individual marker and all-high profiles conferred favorable prognosis. However, when adjusted for sTIL density, stromal and intratumoral lymphocytic subsets lost their prognostic significance, while higher sTIL density conferred up to 15% lower risk for relapse. Independently of sTIL density, higher total CD3+ and CD8+ TILs conferred 35% and 28% lower risk for relapse, respectively. Stromal and intratumoral CD3+, CD8+ and FOXP3+ TIL density do not seem to add prognostic information over the morphologically assessed sTIL density, which is worth introducing in routine histology reports.

Prognostic prediction of apparent diffusion coefficient obtained by diffusion-weighted MRI in mass-forming intrahepatic cholangiocarcinoma.

We evaluated apparent diffusion coefficient (ADC) of diffusion-weighted image MRI as a prognostic factor for mass-forming intrahepatic cholangiocarcinoma (IHCC). We enrolled 26 patients who had undergone hepatic resections for mass-forming-type IHCC in this study, and calculated their mean ADC, using diffusion-weighted image MRI (b: 0, 20, 800seconds/mm2 ; 1.5T MRI). Patients were divided into the ADCHigh and the ADCLow groups at the median ADC value (n=13 for both). We also immunohistochemically evaluated hypoxia-inducible factor (HIF)-1α in tumor tissue. Median age in the ADCLow was older (P=.03), and showed significant higher rate of scirrhous tumor (P=.02). The 5-year overall survival rate in the ADCLow group was significantly worse than in the ADCHigh group (P=.04). In multivariate analysis, hilar tumor, portal vein invasion and low ADC were independent prognostic factors (P<.05). The ADCLow group also had a higher rate of high HIF-1α expression than the ADCHigh group (P<.05). Representative case of ADCLow group showed rich stroma and high HIF-1α expression. The ADC values in MRIs can predict IHCC prognosis, and correlated with stromal density and HIF-1α expression.

Abstract P5-02-13: A novel detection technique for collagen (and other macromolecules) on H&amp;E slides without special stains for improved cancer prognostics

Abstract Background: Stromal density including the characterization of the type, abundance and alignment of collagen and other extracellular matrix (ECM) protein fibers in proximity to breast precancers and cancers has emerged as an important and evolving tool for understanding tumor progression and stratifying/classifying mortality risk. Standard methods for this evaluation including second harmonic generation (SHG) microscopy requires access to fresh or FFPE tissue samples of limited availability. In contrast, standard diagnostic H&amp;E slides are more readily available for large retrospective and prospective studies. Methods: We developed a method for dual emission and transmission (DUET) microscopy that employs combined brightfield and fluorescence modalities, plus quantitative image analysis. The prototype instrument has whole-slide scanning capabilities for collagen detection and pixel-accurate alignment with conventional H&amp;E-stained images. We have analyzed a retrospective cohort of 700 breast cancers with available outcome follow-up of greater than 5 years using this DUET method Results: DUET microscopy provides clear discrimination of collagen and ECM directly from standard H&amp;E specimens, requiring only a few minutes to scan whole slides. Using the method, contrast enhancement similar to special staining techniques (e.g. trichrome, periodic acid Schiff’s, and picrosirius red stains). Image analysis from DUET directly corresponds to SHG data. DUET imaging discriminates between stroma surrounding invasive cancer and benign epithelium. DUET imaging of 700 breast cancers shows outcome correlation. Ongoing multivariate analysis comparing standard measures (grade, tumor infiltrating lymphocytes, tumor phenotype, etc.) to the DUET stromal score will be reported. Conclusions: DUET provides a straightforward, rapid and inexpensive approach for highlighting collagen distribution patterns on existing histology slides. This method should enable current research using improved quantitative tools, including AI-based, for correlation with patient outcomes, by enabling the use of standard and available H&amp;E slides. Citation Format: Farzad Fereidouni, Alexander D. Borowsky, Richard M. Levenson. A novel detection technique for collagen (and other macromolecules) on H&amp;E slides without special stains for improved cancer prognostics [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 P5-02-13.