Peritumoral Stroma Research Articles

Correlation of MMP-9 (Matrix Metalloproteinase 9) and SMA (Smooth Muscle Actin) expression with Basal Cell Carcinoma’s risk of recurrence

Basal Cell Carcinoma (BCC) is the most common cancer in many countries, with its incidence steadily rising. Data from the Indonesian Pathologists Association show that among 1,530 skin cancer cases, BCC was the most frequent (39.93%), followed by Squamous Cell Carcinoma (23%) and Malignant Melanoma (7.9%). According to the 2018 WHO classification, BCC is divided into Low-Risk and High-Risk groups based on the risk of recurrence. BCC invades surrounding tissues through extracellular matrix degradation and enhanced cell motility, driven by the expression of MMP-9 (Matrix Metalloproteinase 9) and SMA (Smooth Muscle Actin). This study aims to evaluate the expression of MMP-9 and SMA in BCC using immunohistochemistry and analyze their correlation with the risk of recurrence. A total of 40 paraffin blocks from BCC patients diagnosed between 2019 and 2021 at Dr. Saiful Anwar General Hospital in Malang, Indonesia, were sampled. The blocks were sectioned, stained with MMP-9 and SMA antibodies, and examined for positive expression indicated by brown staining in the cytoplasm of tumor cell and peritumoral stroma. Spearman correlation showed r = 0.811 for MMP-9 and r = 0.857 for SMA (p < 0.05, CI: 95%), indicating a significant relationship between these markers and the risk of recurrence. Higher MMP-9 and SMA expression corresponded to a higher risk of BCC recurrence, suggesting their potential as prognostic biomarkers. However, further research is needed to assess recurrence risk more accurately and determine the most appropriate therapy.

Relationship of Stromal Tumor Infiltrating Lymphocytes (STILs) with Grading Histopathology in Penis Squamous Cell Carcinoma

ABSTRACT Background:Penile squamous cell carcinoma is the most common penile malignance found in the world. Assessment of stromal tumor infiltrating lymphocytes (STILs) associated with grading against penile squamous cell carcinoma can provide a potential prognostic picture and help determine the action. Method:Cross-sectional analytical study with 32 samples of histopathological preparations diagnosed as penile squamous cell carcinoma at H. Adam Malik Hospital Medan. The assessment of stromal TILs was carried out by assessing the number of lymphocyte inflammatory cells in the peritumoral stroma and then determined the assessment score, zero score no lymphocytes (minimum), score one found 0-10% lymphocytes (moderately), score two found 20-40% lymphocytes (massive) , score three found 50-90% lymphocytes. Grading assessments are categorized into grade I (well differentiated), grade II (moderately differentiated), and grade III (poorly differentiated). Result:The most grading found was grade II (43.8%) and the most STILs found were score three (50.0%). There is a significant relationship between grading and STILs in penile squamous cell carcinoma (p value=0.0001) i.e. the higher the STILs score , the lower the tumor grade. Conclusion:There is a relationship between STILs and histopathological grading in Penile Squamous Cell Carcinoma so that it can be used as an indikasi prognosis in patients with penile squamous cell carcinoma.

Shear-Wave Elastography Gradient Analysis of Newly Diagnosed Breast Tumours: A Critical Analysis.

A better understanding of the peritumoral stroma changes due to tumour invasion using non-invasive diagnostic methods may improve the differentiation between benign and malignant breast lesions. This study aimed to assess the correlation between breast lesion differentiation and intra- and peritumoral shear-wave elastography (SWE) gradients. A total of 135 patients with newly diagnosed breast lesions were included. Intratumoral, subsurface, and three consecutive peritumoral SWE value measurements (with three repetitions) were performed. Intratumoral, interface, and peritumoral gradients (Gradient 1 and Gradient 2) were calculated using averaged SWE values. Statistical analysis included descriptive statistics and an ordinary one-way ANOVA to compare overall and individual gradients among Breast Imaging-Reporting and Data System (BI-RADS) 2, 3, and 5 groups. Malignant tumours showed higher average SWE velocity values at the tumour centre (BI-RADS 2/3: 4.1 ± 1.8 m/s vs. BI-RADS 5: 4.9 ± 2.0 m/s, p = 0.04) and the first peritumoral area (BI-RADS 2/3: 3.4 ± 1.8 m/s vs. BI-RADS 5: 4.3 ± 1.8 m/s, p = 0.003). No significant difference was found between intratumoral gradients (0.03 ± 0.32 m/s vs. 0.0 ± 0.28 m/s; p > 0.999) or gradients across the tumour-tissue interface (-0.17 ± 0.18 m/s vs. -0.13 ± 0.35 m/s; p = 0.202). However, the first peritumoral gradient (-0.16 ± 0.24 m/s vs. -0.35 ± 0.31 m/s; p < 0.0001) and the second peritumoral gradient (-0.11 ± 0.18 m/s vs. -0.22 ± 0.28 m/s; p = 0.037) were significantly steeper in malignant tumours. The AUC was best for PTG1 (0.7358) and PTG2 (0.7039). A threshold value for peritumoral SWI PT1 above 3.76 m/s and for PTG1 below -0.238 m/s·mm-1 indicated malignancy in 90.6% of cases. Evaluating the peritumoral SWE gradient may improve the diagnostic pre-test probability, as malignant tumours showed a significantly steeper curve of the elasticity values in the peritumoral stroma compared to the linear regression with a relatively flat curve of benign lesions.

IR-780 Dye-based Targeting of Cancer-associated Fibroblasts Improves Cancer Immunotherapy by Increasing Intra-tumoral T Lymphocytes Infiltration.

Immune-checkpoint inhibitors (ICIs) against programmed death (PD)-1/PD-L1 pathway immunotherapy have been demonstrated to be effective in only a subset of patients with cancer, while the rest may exhibit low response or may develop drug resistance after initially responding. Previous studies have indicated that extensive collagen-rich stroma secreted by cancer-associated fibroblasts (CAFs) within the tumor microenvironment is one of the key obstructions of the immunotherapy for some tumors by decreasing the infiltrating cytotoxic T cells. However, there is still a lack of effective therapeutic strategies to control the extracellular matrix by targeting CAFs. The enhanced uptake of IR-780 by CAFs was assessed by using in vivo or ex vivo nearinfrared fluorescence imaging, confocal NIR fluorescent imaging, and CAFs isolation testing. The fibrotic phenotype down-regulation effects and in vitro CAFs killing effect of IR-780 were tested by qPCR, western blot, and flow cytometry. The in vivo therapeutic enhancement of anti-PD-L1 by IR-780 was evaluated on EMT6 and MC38 subcutaneous xenograft mice models. IR-780 has been demonstrated to be preferentially taken up by CAFs and accumulate in the mitochondria. Further results identified low-dose IR-780 to downregulate the fibrotic phenotype, while high-dose IR-780 could directly kill both CAFs and EMT6 cells in vitro. Moreover, IR-780 significantly inhibited extracellular matrix (ECM) protein deposition in the peri-tumoral stroma on subcutaneous EMT6 and MC38 xenografts, which increased the proportion of tumor-infiltrating lymphocytes (TILs) in the deep tumor and further promoted anti-PD-L1 therapeutic efficacy. This work provides a unique strategy for the inhibition of ECM protein deposition in the tumor microenvironment by targeted regulating of CAFs, which destroys the T cell barrier and further promotes tumor response to PD-L1 monoclonal antibody. IR-780 has been proposed as a potential therapeutic small-molecule adjuvant to promote the effect of immunotherapy.

Abstract PO1-25-05: Adipose-enriched peri-tumoral stroma prognosticates poorer survival in breast cancers

Abstract Background: Adjuvant breast cancer therapy is informed by whether a tumour is positive or negative for the biomarkers ER, PgR, and HER2, often without regard to level of positivity. Quantitation has been proposed to improve therapeutic management. Adjunctive statistical standardization has been proposed to improve inter-laboratory comparability of biomarkers results. Methods: This primary report utilized adjunctive statistical standardization of machine-quantitated image analysis biomarker assessments. CCTG MA.27 (NCT00066573) is an adjuvant phase III trial of exemestane versus anastrozole in postmenopausal women with ER+ and/or PgR+ tumours. IHC ER, PgR, and HER2 were centrally assessed, with FISH (HER2;HER2/CEP17) determinations for equivocal IHC HER2. HSCOREs were statistically standardized to a mean of 0, standard deviation of 1 following Box-Cox variance stabilization transformations of square for ER and natural logarithm for PgR (0.1 was added to 0 HSCOREs). The primary endpoint was STEEP distant disease-free survival (DDFS) at the longest trial follow-up of median 4.1 years. Survival was described with Kaplan-Meier plots. The univariate Wilcoxon (Peto-Prentice) test statistic was used with usual designation of negative/positive (0; &amp;gt;0), and standardized cut-points at standard deviations about mean of 0(&amp;lt;-1; (-1,0]; (0,1]; &amp;gt;1). Cox multivariate regressions adjusted for age, T and N stage, grade, lymphovascular invasion, treatment, and baseline patient demographics, utilized likelihood ratio tests. Nominal significance was p=0.05. Results: Of the 7576 women accrued, 3048 had machine-quantitated image analysis results: 2900 (95%) for ER; 2726 (89%) for PgR. Only 8 women were ASCO/CAP ER- (HSCORE 0); PgR HSCORE was 0 for 533. Statistically standardized units differentiated DDFS ER levels (p&amp;lt; 0.001) and PgR levels (p&amp;lt; 0.001). In adjusted multivariate analyses, higher ER HSCORE was associated with better DDFS (p=0.05) with weak evidence of an association (p=0.11) for standardized HSCORE, and no significant association (respectively, p=0.28, p=0.54) in models with PgR. Higher PgR was associated with better DDFS (p=0.001) in all multivariate assessments, including those with ER. Conclusions: DDFS was superior for patients with higher ER and PgR standardized units compared with those with HSCOREs &amp;lt;-1. Adjunctive statistical standardization, similar to that mandated for clinical practice by the World Health Organization for BMD, should improve inter-laboratory comparability of biomarker results for similar patient populations. Biomarker N DDFS DDFS 5-year (%) 95% CI ER total 2900 ER &amp;lt;-1 506 86 (82, 91) ER (-1, 0] 934 94 (92, 96) ER ( 0, 1] 919 94 (92, 96) ER 1 541 96 (93, 98) PgR total 2726 PgR &amp;lt;-1 734 89 (86, 92) PgR (-1, 0] 439 92 (89, 95) PgR ( 0, 1] 967 95 (93, 96) PgR 1.0 586 98 (97,100) Citation Format: Hannah Lau, Veronique Kiak Mien Tan, Benita Kiat Tee Tan, Yirong Sim, Jelmar Quist, Aye Aye Thike, Puay Hoon Tan, Shazib Pervaiz, Anita Grigoriadis, Kanaga Sabapathy. Adipose-enriched peri-tumoral stroma prognosticates poorer survival in breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-05.

Peritumoral stroma and systemic inflammatory response in cervical cancer.

To compare cervical stroma in advanced cervical cancer with the control group; to compare, in the pre-treatment period, hemogram parameters in patients with advanced cervical cancer with the same parameters as the control group; and to verify if there is an association of stromal markers with prognostic factors in cervical cancer. We prospectively evaluated 16 patients diagnosed with advanced invasive cervical cancer. A control group of 22 patients was used (uterine leiomyoma). Immunohistochemistry was performed to verify the stromal immunostaining of alpha-smooth muscle actin (SMA) and fibroblast activation protein alpha (FAP). Immunostainings and hemogram parameters were compared using Fisher's exact and Mann-Whitney Test, respectively. Strong FAP immunostaining was more frequent in patients with cervical cancer when compared with patients with leiomyoma (P = 0.0002). Regarding SMA, strong immunostaining was also found more in the group of cancer patients compared to the control group (P &lt; 0.00001). The neutrophil-lymphocyte ratio (NLR) values were higher in the cancer patient group compared to the control group (P = 0.0019). There was no association of the parameters studied with prognostic factors. Strong FAP and SMA immunostaining was found more in patients with cervical cancer when compared to the control group. NLR values were also higher in cervical cancer.

Abstract P64: Adipose-enriched Peri-tumoral Stroma Prognosticates Poorer Survival in Breast Cancers

Abstract Background: Breast cancers display a large degree of diversity between and within tumors. Intra-tumoral heterogeneity is contributed by variations in tumor cells and non-malignant stromal and immune cells of the tumor microenvironment (TME). So far, studies on the TME have focused on intra-tumoral stroma, whereas little attention has been given to the peri-tumoral microenvironment, extending beyond the tumor margins into the surrounding histologically normal-appearing tissues. Here, we hypothesized that intrinsic intra-tumoral heterogeneity impacts the more distal peri-tumoral stroma, which may in turn affect tumor growth and development. Methods: A systematic, multi-regional transcriptomic profiling analysis of the tumor and peri-tumoral regions from 8 ER+/PR+/HER2− invasive breast carcinomas was performed using the human Clariom™ S Assay. For each patient, 5 spatially distinct tumor samples were obtained, while peri-tumoral samples were collected from 4 different sides and at varying distances up to 7 cm from tumor margins. Pathway enrichment analyses and cellular deconvolution were performed to determine their molecular characteristics and cell type compositions. Gene expression signatures identified from peri-tumoral samples were applied to tumor-adjacent normal RNA-seq samples from 50 TCGA ER+/PR+/HER2− breast cancer patients to determine their prognostic value. Results: The 8 tumors displayed varying levels of spatial diversity in their biological properties, including 3 tumors with basal-classified regions. Four peri-tumoral clusters with distinct pathway activities and cell compositions were identified, but were independent of distance and location to the tumor. Two clusters reflect the major breast peri-tumoral states – a pro-inflammatory, adipose-enriched phenotype which was significantly associated with poorer overall survival (HR=4.28, p-value=0.02), and an adaptive immune cell- and myofibroblast-enriched phenotype. Conclusion: This suggests that the peri-tumoral stroma may be an important determinant in cancer progression. Moreover, independent of spatially-defined patterns, the peri-tumoral phenotype appears to be driven by inter-individual variations in the proportion of fibroglandular and fat tissue of the breast. Citation Format: Hannah Si Hui Lau, Veronique Kiak Mien Tan, Benita Kiat Tee Tan, Yirong Sim, Jelmar Quist, Aye Aye Thike, Puay Hoon Tan, Shazib Pervaiz, Anita Grigoriadis, Kanaga Sabapathy. Adipose-enriched Peri-tumoral Stroma Prognosticates Poorer Survival in Breast Cancers [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P64.

Abstract 159: LYVE-1-expressing macrophages modulate the hyaluronan-containing extracellular matrix in the mammary stroma and contribute to mammary tumor growth

Abstract Macrophages represent a heterogeneous myeloid population with diverse functions in normal tissues and tumors. While macrophages expressing the cell surface marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) have been identified in stromal regions of the normal mammary gland and in the peri-tumoral stroma, their functions within these regions are not well-understood. Using a genetic LYVE-1+ macrophage deletion mouse model, we demonstrate that loss of LYVE-1+ macrophages is associated with altered extracellular matrix remodeling in the normal mammary gland and reduced mammary tumor growth in vivo. In further studies focused on investigating the functions of LYVE-1+ macrophages in the tumor microenvironment, we demonstrate that LYVE-1 expression correlates with an increased ability of macrophages to bind, internalize, and degrade hyaluronan. Consistent with this, we show that deletion of LYVE-1+ macrophages correlates with increased hyaluronan accumulation in both the normal mammary gland and in mammary tumors. Analysis of single cell RNA sequencing of macrophages isolated from these tumors reveals that deletion of LYVE-1+ macrophages in tumors drives a shift in the majority of the remaining macrophages towards a pro-inflammatory phenotype, as well as an increase in CD8+ T cell infiltration. Together, these findings demonstrate that LYVE-1+ macrophages represent a tumor-promoting anti-inflammatory subset of macrophages that contributes to hyaluronan remodeling in the tumor microenvironment. Citation Format: Alexis K. Elfstrum, Annisa H. Rumahorbo, Braedan M. McCluskey, Kathryn L. Schwertfeger. LYVE-1-expressing macrophages modulate the hyaluronan-containing extracellular matrix in the mammary stroma and contribute to mammary tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 159.

Abstract 2150: Spatial transcriptomic analysis of histologically matched regions adjacent to and within prostate cancer reveals RNA expression differences between African Americans and European Americans

Abstract Introduction. Prostate cancer (PCa) of American men of African ancestry (AA) is diagnosed at an earlier median age and more advanced stage with a poorer prognosis and significantly higher mortality than in Americans of primarily Northern European ancestry (EA). The mechanism of how ancestry contributes to aggressive PCa in AA patients is not well understood. Given the inherent heterogeneity and multifocality of prostate cancer, Spatial transcriptomics is of particular interest because it allows distinct cell populations within the tumor microenvironment to be characterized, and their possible effects on local tumors to be identified. Here we compare transcription between four AA and four EA patients in regions of peritumoral stroma, inflammation, Benign Prostatic Hyperplasia (BPH), and Gleason score patterns 3 (G3) and 4 (G4). Methods: Formalin fixed, paraffin embedded (FFPE) prostatectomy tissue blocks were obtained from 8 patients (4 AA and 4 EA) prior to treatment of the patients and matched for clinical variables (i.e., biochemical relapse, age, Gleason score, and tumor stage). 10um tissue sections were mounted onto Visium Spatial Gene Expression Slides and processed following 10x Genomics protocol. Sample counts were processed through Space Ranger. 18000 unique genes, spatial barcodes, and clinician guided annotations were imported to the R program Seurat. Data was processed using SCTransform. Significance of each transcript differences between each tissue were calculated. Results: Digital histologic slides of prostate cancer specimens from AA (n=4) and EA (n=4) patients were annotated by a urologic pathologist to identify regions of peritumoral stroma, inflammation, BPH, G3, and G4. Each spot was interrogated for the expression of 18000 transcripts, with an average of 17796 being detected per spot. Among the largest differences observed between AA and EA, G3 and G4 had distinct transcriptome profiles in the four AA as compared to G3 and G4 in four EA. Also, when differential expression (padj &amp;lt; 0.05) in peritumoral stroma was compared to G4 in AA, we identified 290 differentially expressed genes, whereas the same comparison in EA cohorts identified 600 differentially expressed genes. Conclusion: Spatial transcriptomic analysis revealed differences in gene expression across multiple tissue types in prostate cancer patients of different ancestries. Citation Format: Vinay Kumar, Dominic Castaneda, James A. Nguyen, Giovanna A. Giannico, Michael M. Ittmann, Patricia Castro, Omid Yazdanpanah, Arash Rezazadeh, Tara S. Jennings, Michael Mcclelland, Farah Rahmatpanah. Spatial transcriptomic analysis of histologically matched regions adjacent to and within prostate cancer reveals RNA expression differences between African Americans and European Americans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2150.

Insights into the Tumor Microenvironment-Components, Functions and Therapeutics.

Similarly to our healthy organs, the tumor tissue also constitutes an ecosystem. This implies that stromal cells acquire an altered phenotype in tandem with tumor cells, thereby promoting tumor survival. Cancer cells are fueled by abnormal blood vessels, allowing them to develop and proliferate. Tumor-associated fibroblasts adapt their cytokine and chemokine production to the needs of tumor cells and alter the peritumoral stroma by generating more collagen, thereby stiffening the matrix; these processes promote epithelial-mesenchymal transition and tumor cell invasion. Chronic inflammation and the mobilization of pro-tumorigenic inflammatory cells further facilitate tumor expansion. All of these events can impede the effective administration of tumor treatment; so, the successful inhibition of tumorous matrix remodeling could further enhance the success of antitumor therapy. Over the last decade, significant progress has been made with the introduction of novel immunotherapy that targets the inhibitory mechanisms of T cell activation. However, extensive research is also being conducted on the stromal components and other cell types of the tumor microenvironment (TME) that may serve as potential therapeutic targets.

Association between high expression of intratumoral fibroblast activation protein and survival in patients with intrahepatic cholangiocarcinoma

BackgroundCancer-associated fibroblasts (CAFs) have been reported to exhibit protumorigenic effects. Among the well-known CAF markers such as smooth muscle actin (SMA) and fibroblast activation protein (FAP), high expression of SMA in the peritumoral stroma has been reported to be a prognostic factor in various cancers. However, the effect of high FAP expression in intrahepatic cholangiocarcinoma (IHCC) has not been fully clarified. We evaluated the expression of CAF markers, focusing on FAP expression in the peripheral and intratumoral regions, to clarify the association with survival in patients with IHCC.MethodsThe study cohort comprised 37 patients who underwent curative resection for IHCC. The FAP expressions were evaluated in the peripheral and intratumoral regions of the resected tissues. Clinicopathological factors and survival outcomes were investigated between patients with high versus low FAP expression. Uni- and multivariate analyses were performed to identify the prognostic factors for overall survival and relapse-free survival.ResultsThe median area percentages of FAP expression in the peripheral and intratumoral regions were 15.5% and 17.8%, respectively. High FAP expression in the intratumoral region was significantly associated with worse overall survival and disease-free survival than low FAP expression in the intratumoral region. Multivariate analysis identified high intratumoral FAP expression as a risk factor for worse overall survival (hazard ratio, 2.450; p = 0.049) and relapse-free survival (hazard ratio, 2.743; p = 0.034).ConclusionsHigh intratumoral FAP expression was associated with worse survival, suggesting that intratumoral FAP expression represents malignant progression in patients with IHCC.

Adipose-enriched peri-tumoral stroma, in contrast to myofibroblast-enriched stroma, prognosticates poorer survival in breast cancers

Despite our understanding of the genetic basis of intra-tumoral heterogeneity, the role of stromal heterogeneity arising from an altered tumor microenvironment in affecting tumorigenesis is poorly understood. In particular, extensive study on the peri-tumoral stroma in the morphologically normal tissues surrounding the tumor is lacking. Here, we examine the heterogeneity in tumors and peri-tumoral stroma from 8 ER+/PR+/HER2− invasive breast carcinomas, through multi-region transcriptomic profiling by microarray. We describe the regional heterogeneity observed at the intrinsic molecular subtype, pathway enrichment, and cell type composition levels within each tumor and its peri-tumoral region, up to 7 cm from the tumor margins. Moreover, we identify a pro-inflammatory adipose-enriched peri-tumoral subtype which was significantly associated with poorer overall survival in breast cancer patients, in contrast to an adaptive immune cell- and myofibroblast-enriched subtype. These data together suggest that peri-tumoral heterogeneity may be an important determinant of the evolution and treatment of breast cancers.

OVER SERÖZ KARSİNOMUNDA VE BORDERLİNE SERÖZ TÜMÖRDE İMMÜNHİSTOKİMYASAL PINCH/LIMS-1 ANTİKOR EKSPRESYONUNUN KLİNİKOPATOLOJİK DEĞERİ

Objective&#x0D; Particularly interesting cys-his rich protein (PINCH/&#x0D; LIMS-1), a protein implicated in cell adhesion, is&#x0D; assumed to oversee the development and invasion of&#x0D; cancer cells in tumors and tumor-associated stroma.&#x0D; This study aimed to assess PINCH-1 expression in&#x0D; serous borderline tumor (SBT) and serous carcinoma&#x0D; (SC) in the tumor and peritumoral stroma and&#x0D; scrutinize any associations between its expression&#x0D; and various clinical and pathological parameters.&#x0D; Material and Method&#x0D; In this study, the expression of the PINCH-1 antibody&#x0D; was analyzed in 21 cases of SBT and 89 cases of&#x0D; SC using the indirect immunoperoxidase technique&#x0D; with streptavidin/HRP-biotin. The staining pattern of&#x0D; PINCH-1 in the tumor and peritumoral stroma was&#x0D; evaluated using a semiquantitative scoring method.&#x0D; The staining procedure used in the study allowed for&#x0D; the accurate identification of PINCH-1 expression,&#x0D; and the data obtained through the semiquantitative&#x0D; scoring method provided a reliable of assessing&#x0D; the degree and intensity of PINCH-1 staining. Thus,&#x0D; the correlation between PINCH-1 expression and&#x0D; various pathologic factors such as patient age, tumor&#x0D; size, FIGO stage, intra-abdominal washing cytology,&#x0D; capsule invasion, tumor location in the ovary, tumor&#x0D; grade, and cancer antigen 125 (CA125) levels at the&#x0D; time of diagnosis was examined.&#x0D; Results&#x0D; The study found that PINCH-1 was more prevalent&#x0D; in cases of SC than in SBT cases. The tumors in SC&#x0D; cases had stronger staining than those in SBT cases&#x0D; (p

Usefulness of COL11A1 as a Prognostic Marker of Tumor Infiltration.

Determining the infiltration of carcinomas is essential for the proper follow-up and treatment of cancer patients. However, it continues to be a diagnostic challenge for pathologists in multiple types of tumors. In previous studies (carried out in surgical specimens), the protein COL11A1 has been postulated as an infiltration marker mainly expressed in the extracellular matrix (ECM). We hypothesized that a differential expression of COL11A1 may exist in the peritumoral stroma of tumors that have acquired infiltrating properties and that it may be detected in the small biopsies usually available in normal clinical practice. In our study, we performed immunohistochemical staining in more than 350 invasive and noninvasive small samples obtained via core needle biopsy (CNB), colonoscopy, or transurethral resection of bladder tumor (TURBT) of breast, colorectal, bladder, and ovarian cancer. Our results revealed that COL11A1 immunostaining had a sensitivity to classify the samples into infiltrative vs. noninfiltrative tumors of 94% (breast), 97% (colorectal), >90% (bladder), and 74% (ovarian); and a specificity of 97% (breast), 100% (colorectal), and >90% (bladder). In ovarian cancer, the negative predictive value (0.59) did not present improvement over the usual histopathological markers. In all samples tested, the cumulative sensitivity was 86% and the specificity 96% (p < 0.0001). COL11A1-positive immunostaining in small biopsies of breast, colon, bladder and ovarian cancer is an accurate predictive marker of tumor infiltration that can be easily implemented in daily clinical practice.

The Peritumoral CD8+/FOXP3+ Cell Ratio Has Prognostic Value in Triple-negative Breast Cancer.

Compelling data has demonstrated the prognostic significance of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC), a subtype generally associated with a poor clinical outcome but highly heterogeneous in nature. There have been limited studies investigating the importance of subsets of T cells in TILs. Further, the significance of intratumoral versus peritumoral TILs remains controversial. We examined the prognostic value of tumor-associated CD8+ cytotoxic T cells and FOXP3+ regulatory T cells in 35 chemotherapy-naive TNBC cases with a tumor-host interface in the tissue sections. The CD8+ and FOXP3+ cell count was expressed by immunoreactive cells per high-power field in an average of 10 high-power fields. There was a wide range of CD8+ and FOXP3+ T cells within the peritumoral and intratumoral stroma. Both CD8+ and FOXP3+ TILs were significantly higher at the former location as compared with the latter (P<0.0001 and 0.003, respectively). The numbers of CD8+ and FOXP3+ T cells, either within peritumoral or intratumoral stroma, were not significantly associated with distant relapse-free or disease-specific survival. However, the peritumoral CD8+/FOXP3+ ratio of TILs was significantly associated with prolonged relapse-free survival (P=0.04) and disease-specific survival (P=0.02). This association was not observed with the CD8+/FOXP3+ ratio of intratumoral TILs. These observations suggest that the immunologic balance in the tumor microenvironment might determine antitumor immunity. Further, the peritumoral TILs appear to play a more important role in the progression of TNBC when compared with the intratumoral TILs, thus reaffirming the necessity of revisiting the method for the assessment of TILs.

010 Enhanced outcome prediction in cutaneous squamous cell carcinoma using deep-learning and computational histopathology (cSCCnet)

Abstract Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer and leads to a significant disease burden. Although metastases occur in &amp;lt; 5% of patients, effective treatments are limited and 5-year survival is poor, with mortality rates equivalent to those of melanoma in some regions of the world. Despite recent advances in understanding the molecular pathogenesis of cSCC, patient risk stratification is limited and based on clinicopathological staging, with few validated prognostic biomarkers available to guide management [de Jong E, Lammerts MUPA, Genders RE, Bowes Bavinck JN. Update of advanced cutaneous squamous cell carcinoma. J Eur Acad Dermatol Venereol 2022; 36(Suppl. 1):6–10]. In this study, we aimed to use artificial intelligence technology to develop a deep learning model using digitized whole-slide images of cSCC to quantify the risk of metastasis. Routine haematoxylin and eosin-stained slides of 256 primary cSCCs from immunocompetent patients at four collaborating centres were scanned. The area of interest encompassing tumour and peritumoral stroma was annotated, and 512 × 512 pixel tiles were generated from each slide. High-quality tiles (containing sufficient tumour content) were pooled together, with patient-level labels inherited by tiles. We trained multiple models using an 80:10:10 split for training, validation and testing, and the model achieving the highest tile- and slide-level validation accuracy was identified (ResNet50). The final model, namely, cSCCnet, achieved 88% sensitivity, 73% specificity, 64% positive predictive value and 92% negative predictive values on the independent test set. Blinded review of the top predictive tiles by three histopathologists suggested that the predictive features identified by cSCCnet were distinct from histopathological features commonly used by clinicians to estimate metastatic risk. We have demonstrated that it is possible to train a deep learning model using digital pathology to predict the risk of metastasis. cSCCnet surpassed the published performance of currently used staging systems, as well as commercially available gene panel testing (de Jong et al.; Wysong A, Newman JG, Covington KR et al. Validation of a 40-gene expression profile test to predict metastatic risk in localized high-risk cutaneous squamous cell carcinoma. J Am Acad Dermatol 2021; 84:361–9). The morphological features and patterns identified by cSCCnet are different from established histopathological high-risk criteria, indicating that this technology can enhance current practice. Although further validation in a prospective cohort is pending, this is an important step toward creating a risk stratification tool that can be incorporated into routine pathology workflows, significantly improving patient outcomes for this common cancer.

Fiber density and matrix stiffness modulate distinct cell migration modes in a 3D stroma mimetic composite hydrogel.

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode -resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment and understanding of the relationships between matrix attributes and cell migration mode can aid ongoing efforts to identify effective cancer therapeutics that address cell plasticity-based therapy resistances. STATEMENT OF SIGNIFICANCE: Stromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber-reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of migration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.

Assessment of tumoral and peritumoral inflammatory reaction in cutaneous malignant melanomas.

Skin cancer is one of the most common types of cancer, with an increasing worldwide incidence in recent decades. The main risk factor for increasing the skin cancer incidence is ultraviolet (UV) radiation. Of the two major forms of skin cancer (melanomas and non-melanotic cancers), the cutaneous melanoma (CM) is the most aggressive form, causing about 80% of the deaths resulted from this type of tumor. Malignant melanoma develops through malignant transformation of melanocytes in the skin because of prolonged exposure to solar or artificial UV. The malignant transformation of the melanocytes in the skin is accompanied by the presence of a local inflammatory reaction that, in the initial stages of carcinogenesis, would oppose to tumor development. Chronic exposure to UV or other etiopathogenic factors induces chronic inflammation, which, by producing inflammatory molecules (cytokines, chemokines, prostaglandins), constitutes a tumoral microenvironment that favors carcinogenesis, tumor invasion, metastasis, and the presence of neoplastic "mutant cells" that avoid the protective action of the immune system. Using immunohistochemistry techniques, we assessed the intra- and peritumoral inflammatory infiltrate cells in CM. The chronic inflammatory infiltrate presented more intense in the peritumoral stroma compared to the intratumoral one, heterogenous, more intensely composed of lymphocytes, plasma cells, macrophages, and mast cells (MCs), the most numerous cells in the inflammatory infiltrate being T-lymphocytes, plasma cells and macrophages; B-lymphocytes and MCs were in a small number, especially intratumorally. Inflammatory cells had a direct contact with tumor cells, blood vessels, connective matrix, suggesting that the inflammatory microenvironment plays an important role in carcinogenesis, tumor invasion, local angiogenesis, and tumor metastasis.

Abstract 2108: High prevalence of immune exclusion in cancer as determined by pathologist assessment and image analysis

Abstract Background: Immune infiltrated tumors have high levels of lymphocytes contacting tumor cells and are more responsive to checkpoint inhibitors. Tumors with few lymphocytes in contact with tumor cells can be divided into desert or excluded phenotypes based on lymphocyte absence/paucity or restriction to the peritumoral stroma, respectively. Standard methods to systematically identify and characterize immune exclusion for patient stratification are lacking. Methods: Slides from colorectal (CRC), non-small cell lung (NSCLC), ovarian (OC), pancreatic (PDAC), and triple-negative breast cancers (TNBC), and leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS) were stained with multiplex IHC (mIHC) for CD8 and panCK. Pathologist assessment (PA) was done to classify the tumors as: desert, with a paucity of CD8 T cells; excluded, with CD8 T cells not penetrating the tumor parenchyma; and infiltrated, with CD8 T cells within the tumor parenchyma. For the carcinomas, adjacent sections were stained with a multiplex immunofluorescence (mIF) panel containing CD8 and a tumor cell marker. Image analysis (IA) was performed on the mIF images to quantify CD8 cell density in the tumor parenchyma and stroma to categorize immune phenotypes. Results: Immune phenotypes were classified for 143 samples based on PA of mIHC images and 103 samples by IA of mIF images (see Table 1). Immune exclusion as determined by both PA and IA was highest in CRC, PDAC, and TNBC. IA differed from PA in 25 (24.3%) cases. Pathologist review of the discordant cases revealed discrepancies were generally due to tumor heterogeneity, thresholding, assessment of cells at the tumor-stroma boundaries, necrosis, and artifacts. Conclusion: Immune exclusion is highly prevalent in the examined carcinoma types. IA-based approaches, guided by pathologist input, offer promise to quantitatively determine tumor immune phenotypes in a quick and systematic way to guide patients to the most effective therapy. Table 1. Pathologist Assessment Classification Image Analysis Classification Tumor Type n Desert (%) Excluded (%) Infiltrated % Desert (%) Excluded (%) Infiltrated % CRC 20 3 (15.0) 14 (70.0) 3 (15.0) 6 (30.0) 11 (55.0) 3 (15.0) NSCLC 21 6 (28.6) 12 (57.1) 3 (14.3) 6 (28.6) 9 (42.9) 6 (28.6) OC 20 3 (15.0) 9 (45.0) 8 (40.0) 9 (45.0) 3 (15.0) 8 (40.0) PDAC 21 6 (28.6) 14 (66.7) 1 (4.8) 7 (33.3) 12 (57.1) 2 (9.5) TNBC 21 3 (14.3) 15 (71.4) 3 (14.3) 4 (19.0) 10 (47.6) 7 (33.3) LMS 20 3 (15.0) 1 (5.0) 16 (80.0) NA NA NA UPS 20 4 (20.0) 1 (5.0) 15 (75.0) NA NA NA Total 143/103 28 (19.6) 66 (46.2) 49 (34.3) 32 (31.1) 45 (43.7) 26 (25.2) Citation Format: Florent Peyraud, Fredrick D. Gootkind, Antoine Italiano, Alban Bessede, Jean-Philippe Guegan, Xinwei Sher, Thomas Schürpf, Guy T. Clifton, Laura A. Dillon. High prevalence of immune exclusion in cancer as determined by pathologist assessment and image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2108.

Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement.

Cells tune adherens junction dynamics to regulate epithelial integrity in diverse (patho)physiological processes, including cancer metastasis. We hypothesized that the spatially confining architecture of peritumor stroma promotes metastatic cell dissemination by remodeling cell-cell adhesive interactions. By combining microfluidics with live-cell imaging, FLIM/FRET biosensors, and optogenetic tools, we show that confinement induces leader cell dissociation from cohesive ensembles. Cell dissociation is triggered by myosin IIA (MIIA) dismantling of E-cadherin cell-cell junctions, as recapitulated by a mathematical model. Elevated MIIA contractility is controlled by RhoA/ROCK activation, which requires distinct guanine nucleotide exchange factors (GEFs). Confinement activates RhoA via nucleocytoplasmic shuttling of the cytokinesis-regulatory proteins RacGAP1 and Ect2 and increased microtubule dynamics, which results in the release of active GEF-H1. Thus, confining microenvironments are sufficient to induce cell dissemination from primary tumors by remodeling E-cadherin cell junctions via the interplay of microtubules, nuclear trafficking, and RhoA/ROCK/MIIA pathway and not by down-regulating E-cadherin expression.