A Deep Learning-Based Assessment Pipeline for Intraepithelial and Stromal Tumor-Infiltrating Lymphocytes in High-Grade Serous Ovarian Carcinoma.

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in patients with epithelial ovarian cancer. However, TIL evaluation has not been used in routine clinical practice because of reproducibility issues. The current study developed two convolutional neural network models to detect TILs and to determine their spatial location in whole slide images, and established a spatial assessment pipeline to objectively quantify intraepithelial and stromal TILs in patients with high-grade serous ovarian carcinoma. The predictions of the established models showed a significant positive correlation with the number of CD8+ T cells and immune gene expressions. Patients with a higher density of intraepithelial TILs had a significantly prolonged overall survival and progression-free survival in multiple cohorts. On the basis of the density of intraepithelial and stromal TILs, patients were classified into three immunophenotypes: immune inflamed, excluded, and desert. The immune-desert subgroup showed the worst prognosis. Gene expression analysis showed that the immune-desert subgroup had lower immune cytolytic activity and T-cell-inflamed gene-expression profile scores, whereas the immune-excluded subgroup had higher expression of interferon-γ and programmed death 1 receptor signaling pathway. The established evaluation method provided detailed and comprehensive quantification of intraepithelial and stromal TILs throughout hematoxylin and eosin-stained slides. It has potential for clinical application for personalized treatment of patients with ovarian cancer.

Interobserver variability in the assessment of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative invasive breast carcinoma influences the association with pathological complete response: the IVITA study.

High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland-Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from -0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the 'ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

Assessment of Lymph Node Stromal Cells as an Underlying Factor in Age-Related Immune Impairment.

Aging negatively impacts immunity, resulting in inefficient responses to vaccinations and infections. Fibroblastic reticular cells (FRCs) are the major stromal cell subset in lymph nodes (LNs) and play an intricate role in the orchestration and control of adaptive immune responses. Although stromal cells have a major impact on immune responses, the impact of aging on LN stromal cells remains unclear. Quantitative analysis of LN stromal cells by flow cytometry revealed that there are no significant differences in the number of stromal cells in young and aged LN at steady state but after influenza infection aged FRCs have delayed expansion as a result of reduced proliferation. Aged LNs also produce reduced levels of homeostatic chemokines, which correlates with reduced homing of naive T cells. Image analysis reveals that young and aged T-cell zone FRCs have similar morphology at steady state and after infection. Furthermore, aged FRCs did not appear to be a contributing factor in the reduced proliferation of young T cells transferred into aged LNs after influenza infection. These results demonstrate that aging alters LN stromal cell response to challenge and these age-related changes may be an underlying contributor to impaired immune responses in the elderly people.

Assessment of presence and characteristics of multipotent stromal cells in human endometrium and decidua.

This review discusses the presence and characteristics of multipotent stromal cells in human endometrium and decidua. A number of research groups have reported the isolation and characterization of multipotent stromal cells from the basal layer of the endometrium, and in a single case just from the menstrual blood, i.e. the superficial functional layer. Similarly, multipotent pre-decidual stromal cells are isolated from early decidua and characterized accordingly. Multipotent endometrial stromal cells and multipotent decidual stromal cells are shown to express the basic features of adult stem cells, which are clonogenicity, self-renewal, a potential to differentiate into adipogenic, osteogenic, chrondrogenic, endothelial-like cells and a specific set of surface molecules (CD73, CD90 and CD105). So far, it is not clear whether the same population of multipotent stromal cells is isolated from the basal endometrium or early decidua because it has been shown that in some cases the differentiation potential of endometrial stromal cells is more restricted in comparison to the decidual stromal cells. It is reasonable to assume that it is one cell population under different control by hormonal, paracrine and autocrine factors. Thus far, the functions of these cells have not been convincingly revealed.

The quantitative assessment of MHC II on thymic epithelium: implications in cortical thymocyte development.

The dynamics of MHC II expression in various thymic stromal compartments was investigated. By including MHC II in flow cytometry in addition to the cortical CDR1, medullary UEA-1 and pan-epithelial G8.8 markers, thymic stromal compartments were subdivided into at least six different populations. The total level of surface and cytoplasmic MHC II from fresh cortical thymic epithelial cells (cTECs) of normal mouse was as high as MHC II levels in medullary thymic epithelial cells (mTECs). MHC II levels as well as the percentages and cycling status of thymic epithelial cell populations expressing MHC II were not static during post-natal development, suggesting quantitative flexibility in presenting signals to the developing thymocytes. Although there was no evidence found for regulation of surface MHC II levels by TCR or by IFN-gamma, the absence of class II transactivator reduced both the level of MHC II expression and the number of MHC II+ cells. Surprisingly, MHC II molecules were found to form distinct focal aggregates on the surface of cTEC but not mTEC using high-resolution analysis by confocal microscopy. Moreover, these aggregates were formed independent of TCR or TCR-bearing cells in the thymus. These aggregates could potentially generate a functional unit containing a much higher local MHC II concentration to yield a higher avidity interaction. We discuss possible mechanisms for positive selection by weak interactions in the presence of such preformed MHC II aggregate units in cTEC.

Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with autoimmune cytopenias.

To investigate whether bone marrow (BM) stem cell compartment and/or BM microenvironment are affected by the immune insult in autoimmune cytopenias (AICs), BM stem cell reserve and function and BM stromal function were studied in 15 AIC patients. Stem cells were evaluated by means of flow cytometry, clonogenic progenitor cell assays, long-term BM cultures (LTBMCs), and limiting dilution assay for quantification of long-term-culture initiating cells (LTC-ICs). Stromal cell function was assessed with the use of preformed irradiated LTBMCs from patients and normal controls, recharged with normal CD34(+) cells. AIC patients exhibited a high number of CD34(+), CD34(+)/CD38(+), and CD34(+)/CD38(-) cells; high frequency of granulocyte-macrophage colony forming units in the BM mononuclear cell fraction; high colony recovery in LTBMCs; and normal LTC-IC frequency. Patient BM stromal layers displayed normal hematopoietic-supporting capacity and increased production of granulocyte-colony stimulating factor. Data from this study support the concept that AIC patients with severe, resistant disease might be appropriate candidates for autologous stem cell transplantation.

Immunohistochemical assessment of prostaglandin H-synthase in bovine endometrial biopsy samples collected throughout the oestrous cycle.

The study was designed to determine the distribution of prostaglandin H-synthase (PGS) also known as cyclooxygenase in specific uterine cell populations during the oestrous cycle. Endometrial biopsy samples were obtained from a total of 10 clinically healthy cows at days 1 (initiation of behavioural oestrus), 8, 15, and 19 of the oestrous cycle. All animals conceived after biopsy regimen. Data of semiquantitatively scored immunoreactivities were analysed using analyses of variance, t-tests for paired data and correlation analyses. Biotin-streptavidin-peroxidase immunostaining technique was employed to localise PGS. Specific staining was consistently present in endothelial cells of arteries but not capillaries and venules. A gradient of staining intensity was clearly apparent within the endometrium: surface epithelial cells and stromal cells located near the endometrial surface are consistently stained more intensely than glandular epithelial cells and stromal cells lying deeper in the endometrium. Days of oestrous cycle also influenced PGS immunoreactivities. Generally, higher immunoreactivities were recorded in surface epithelium, uterine glands and endometrial stromal cells at cycle days 1 and 19 as compared to cycle days 8 and 15. Minimal scoring values were mainly found at cycle day 8. The results of the present study suggest that the amount of bovine endometrial PGS varies considerably with the day of cycle in the above mentioned cell-type- and location-restricted manner. Therefore, the capacity of the bovine uterine mucosa for prostaglandin production may--amongst other factors--depend on the cycle-restricted availability of the respective enzyme systems.

Quantitative and qualitative assessment of CA-125 production by human endometrial epithelial cells: comparison of eutopic and heterotopic epithelial cells.

We have established an in vitro system for the culture of epithelial cells (ECs) from human uterine endometrium to examine the production of CA 125 and to characterize the CA-125 antigen purified from the conditioned media. CA-125 secretion was higher in heterotopic ECs than in eutopic ECs and it was more significant after heterotopic ECs reached confluence than during the logarithmic growth phase. CA-125 expression was observed mainly in the G0/G1-phase. CA-125 expression on cell membranes did not correlate with the volume of CA 125 released per cell, and there was no amplification of CA-125 expression in heterotopic EC membranes. Treatment of the purified CA-125 antigen with 6 M urea yielded a much lower molecular-mass peak (200 kDa). The results of Western blot indicated the presence of a single polydisperse band of 200 kDa in the conditioned media from eutopic ECs, whereas 2 major CA-125 isoforms of 200 kDa and 110 kDa, as well as 2 minor forms of 100 kDa and 70 kDa, were observed in the conditioned media of the heterotopic ECs. We conclude that, in heterotopic ECs, the 110-kDa CA-125 is more prominent than the 200-kDa antigen, and that the elevation of CA-125 levels in the conditioned media could be attributed to significantly increased release of 110-kDa CA-125 from heterotopic ECs.