Tumor-associated immune cell infiltrate density in penile squamous cell carcinomas

Penile squamous cell carcinomas are rare tumor entities throughout Europe. Early lymphonodal spread urges for aggressive therapeutic approaches in advanced tumor stages. Therefore, understanding tumor biology and its microenvironment and correlation with known survival data is of substantial interest in order to establish treatment strategies adapted to the individual patient. Fifty-five therapy naïve squamous cell carcinomas, age range between 41 and 85 years with known clinicopathological data, were investigated with the use of tissue microarrays (TMA) regarding the tumor-associated immune cell infiltrate density (ICID). Slides were stained with antibodies against CD3, CD8 and CD20. An image analysis software was applied for evaluation. Data were correlated with clinicopathological characteristics and overall survival. There was a significant increase of ICID in squamous cell carcinomas of the penis in relation to tumor adjacent physiological tissue. Higher CD3-positive ICID was significantly associated with lower tumor stage in our cohort. The ICID was not associated with overall survival. Our data sharpens the view on tumor-associated immune cell infiltrate in penile squamous cell carcinomas with an unbiased digital and automated cell count. Further investigations on the immune cell infiltrate and its prognostic and possible therapeutic impact are needed.

The effect of clozapine and MIS416 on demyelination, remyelination and immunomodulation in the murine cuprizone model

<p>Multiple Sclerosis (MS) is a disorder of the central nervous system that affects approximately 2.5 million people worldwide. Due to the heterogeneous nature of the disease, and the want of an identified cause, treatment of MS remains difficult. Treatments are available, however these are limited in efficacy and are not suitable for all forms of MS. Disease pathology is characterised by the formation of demyelinating lesions in the central nervous system (CNS) which lead to cognitive and motor impairments associated with the disease. These CNS lesions can be classified as those with immune cell involvement or those without immune cell infiltrate, which are more commonly seen in progressive forms of MS, and currently, there are no treatments available for progressive MS. Due to the limited options available for treating progressive MS, this thesis aims to identify the therapeutic effect provided by the immunomodulatory compounds, MIS416 and clozapine, in a non-immune mediated model of MS, which is believed to more closely resemble progressive disease. Both of these compounds have been shown previously to reduce disease burden in an immune-driven animal model of MS. To investigate the effect of immune-modulating therapies on lesions without immune cell infiltrate, the cuprizone model of non-immune demyelination was used. In summary, the work presented in this thesis found that treatment with MIS416 and clozapine led to improved performance on behavioural assays, although neither agent inhibited cuprizone-induced demyelination or enhanced remyelination. The cellular mechanism behind the observed behavioural improvement is yet to be confirmed. MIS416 was able to maintain its previously identified immunomodulatory properties when administered in this novel setting. Moreover, novel changes to serum growth factors were identified that could provide unexpected benefit to MS patients administered MIS416. In addition to reversing cuprizone-induced behavioural deficits, clozapine reduced LPS-driven inflammatory cytokine production by microglia, indicating that clozapine has the ability to directly reduce inflammation, which may benefit progressive MS patients. Protective effects provided by either of these compounds could aid in the development of unique combination therapies to target both the inflammatory immune component and the cellular components seen at different stages of MS. MIS416 induced changes to serum cytokines and growth factors in the periphery could be harnessed to treat not just MS but other auto-immune diseases characterised by a similar cytokine profile.</p>

The effect of clozapine and MIS416 on demyelination, remyelination and immunomodulation in the murine cuprizone model

<p>Multiple Sclerosis (MS) is a disorder of the central nervous system that affects approximately 2.5 million people worldwide. Due to the heterogeneous nature of the disease, and the want of an identified cause, treatment of MS remains difficult. Treatments are available, however these are limited in efficacy and are not suitable for all forms of MS. Disease pathology is characterised by the formation of demyelinating lesions in the central nervous system (CNS) which lead to cognitive and motor impairments associated with the disease. These CNS lesions can be classified as those with immune cell involvement or those without immune cell infiltrate, which are more commonly seen in progressive forms of MS, and currently, there are no treatments available for progressive MS. Due to the limited options available for treating progressive MS, this thesis aims to identify the therapeutic effect provided by the immunomodulatory compounds, MIS416 and clozapine, in a non-immune mediated model of MS, which is believed to more closely resemble progressive disease. Both of these compounds have been shown previously to reduce disease burden in an immune-driven animal model of MS. To investigate the effect of immune-modulating therapies on lesions without immune cell infiltrate, the cuprizone model of non-immune demyelination was used. In summary, the work presented in this thesis found that treatment with MIS416 and clozapine led to improved performance on behavioural assays, although neither agent inhibited cuprizone-induced demyelination or enhanced remyelination. The cellular mechanism behind the observed behavioural improvement is yet to be confirmed. MIS416 was able to maintain its previously identified immunomodulatory properties when administered in this novel setting. Moreover, novel changes to serum growth factors were identified that could provide unexpected benefit to MS patients administered MIS416. In addition to reversing cuprizone-induced behavioural deficits, clozapine reduced LPS-driven inflammatory cytokine production by microglia, indicating that clozapine has the ability to directly reduce inflammation, which may benefit progressive MS patients. Protective effects provided by either of these compounds could aid in the development of unique combination therapies to target both the inflammatory immune component and the cellular components seen at different stages of MS. MIS416 induced changes to serum cytokines and growth factors in the periphery could be harnessed to treat not just MS but other auto-immune diseases characterised by a similar cytokine profile.</p>

The peri- and intratumoral immune cell infiltrate and PD-L1 status in invasive squamous cell carcinomas of the penis

Introduction Penile carcinomas are rare tumors throughout Europe. Therefore, little attention is drawn to this disease. That makes it important to study tumor-associated key metrics and relate these to known data on penile neoplasias. Materials and methods A cohort of 60 well-defined penile invasive carcinomas with known human papillomavirus (HPV) infection status was investigated. Data on tumor type, grading and staging were recorded. Additionally, data on the peri- and intratumoral immune cell infiltrate in a semiquanititave manner applying an HE stain were assessed. Results Our study showed a significant correlation of immune cell infiltrate and pT stage with overall survival. Therefore, in a subset of tumors, PD-L1 staining was applied. For tumor proportion score (TPS), 26 of 30 samples (87%) were scored >0%. For the immune cell score (IC), 28 of 30 samples (93%) were defined as >0% and for CPS, 29 of 30 samples (97%) scored >0. PD-L1 expression was not associated with overall survival. Conclusion PD-L1 is expressed in penile carcinomas, providing a rationale for targeted therapy with checkpoint inhibitors. We were able to show that immune reaction appears to be prognostically relevant. These data enhance the need for further studies on the immune cell infiltrate in penile neoplasias and show that PD-L1 expression is existent in our cohort, which may be a potential target for checkpoint inhibitor therapy.

Quantitative Assessment of the Immune Microenvironment in Patients With Iatrogenic Laryngotracheal Stenosis

Objective Iatrogenic laryngotracheal stenosis (iLTS) is characterized by fibroinflammatory narrowing of the upper airway and is most commonly caused by intubation injury. Evidence suggests a key role for CD4 T cells in its pathogenesis. The objective of this study is to validate emerging multiplex immunofluorescence (mIF) technology for use in the larynx and trachea while quantitatively characterizing the immune cell infiltrate in iLTS. In addition to analyzing previously unstudied immune cell subsets, this study aims to validate previously observed elevations in the immune checkpoint PD-1 and its ligand PD-L1 while exploring their spatial and cellular distributions in the iLTS microenvironment. Study Design Controlled ex vivo cohort study. Setting Tertiary care center. Methods mIF staining was performed with formalin-fixed, paraffin-embedded slides from 10 patients with iLTS who underwent cricotracheal resection and 10 control specimens derived from rapid autopsy for CD4, CD8, CD20, FoxP3, PD-1, PD-L1, and cytokeratin. Results There was greater infiltration of CD4+ T cells, CD8+ T cells, CD20+ B cells, FoxP3+CD4+ Tregs, and FoxP3+CD8+ early effector T cells in the submucosa of iLTS specimens as compared with controls ( P < .05 for all). PD-1 was primarily expressed on T cells and PD-L1 predominantly on CD4+ cells and “other” cells. Conclusion This study leverages the power of mIF to quantify the iLTS immune infiltrate in greater detail. It confirms the highly inflammatory nature of iLTS, with CD4+ cells dominating the immune cell infiltrate; it further characterizes the cellular and spatial distribution of PD-1 and PD-L1; and it identifies novel immunologic targets in iLTS.

M2 Macrophages Infiltrating Epithelial Ovarian Cancer Express MDR1: A Feature That May Account for the Poor Prognosis

Multi drug resistance protein 1 (MDR1) expression on tumor cells has been widely investigated in context of drug resistance. However, the role of MDR1 on the immune cell infiltrate of solid tumors remains unknown. The aim of this study was to analyze the prognostic significance of a MDR1+ immune cell infiltrate in epithelial ovarian cancer (EOC) and to identify the MDR1+ leucocyte subpopulation. MDR1 expression was analyzed by immunohistochemistry in 156 EOC samples. In addition to MDR1+ cancer cells, we detected a MDR1+ leucocyte infiltrate (high infiltrate >4 leucocytes per field of view). Correlations and survival analyses were calculated. To identify immune cell subpopulations immunofluorescence double staining was performed. The MDR1+ leucocyte infiltrate was associated with human epidermal growth factor receptor 2 (HER2) (cc = 0.258, p = 0.005) and tumor-associated mucin 1 (TA-MUC1) (cc = 0.202, p = 0.022) expression on cancer cells. A high MDR1+ leucocyte infiltrate was associated with impaired survival, especially in patients whose carcinoma showed either serous histology (median OS 28.80 vs. 50.64 months, p = 0.027, n = 91) or TA-MUC1 expression (median OS 30.60 vs. 63.36 months, p = 0.015, n = 110). Similar findings for PFS suggest an influence of MDR1+ immune cells on the development of chemoresistance. A Cox regression analysis confirmed the independency of a high MDR1+ leucocyte infiltrate as prognostic factor. M2 macrophages were identified as main part of the MDR1+ leucocyte infiltrate expressing MDR1 as well as the M2 marker CD163 and the pan-macrophage marker CD68. Infiltration of MDR1+ leucocytes, mostly M2 macrophages, is associated with poor prognosis of EOC patients. Further understanding of the interaction of M2 macrophages, MDR1 and TA-MUC1 appears to be a key aspect to overcome chemoresistance in ovarian cancer.

SUN-LB132 Frequency and Impact of Mutations in Tumor Suppressor Gene CDC73 in General Population and Malignant Tumors

Background: CDC73 gene is a putative tumor suppressor gene, and somatic and germline mutations of this gene have been linked with parathyroid tumors. There are also some reports suggesting that germline CDC73 mutations may increase risk of jaw, kidney and uterine tumors. However, the incidence and significance of CDC73 somatic mutations is largely unknown in tumors other than parathyroid tumors. Methods/Design: 1) Assess CDC73 germline mutation frequency and genotypes in general population using ExAC (Exome Aggregation Consortium) and gnomAD (Genome Aggregation Database); 2) Assess CDC73 somatic mutation frequency in various type of tumors using the Cancer Genome Atlas (TCGA) and non-TCGA datasets; 3) Examine significance of CDC73 mutation in uterine endometrial carcinoma (UCEC). Overall survival, CDC73 mutation signature, gene expression profile (GEP) and tumor immune microenvironment were compared between the UCEC samples with and without CDC73 mutations. Results: In ExAC and gnomAD databases, most of CDC73 germline mutations were mapped to the intron regions of the CDC73 gene and nonsynonymous CDC73 mutations are very rare, with allele frequency ranging from 8.24e-06 to 9.92e-05 and 3.97e-06 to 7.43e-05, respectively. Missense mutations accounted for more than 95% of the nonsynonymous mutations in the CDC73 gene in both databases. 2) In 155 cancer genomics studies including both TCGA and non-TCGA datasets with no overlapping samples, CDC73 was mutated in 0.7% cases and missense mutations comprised 78% of the CDC73 mutations. CDC73 somatic mutations were undetected or rarely detected in endocrine tumors except for parathyroid tumors. 3) UCEC is the tumor type that has the second highest CDC73 somatic mutation rate (8%) after parathyroid tumors. The UCEC with CDC73 mutations had a significantly better overall survival with a logrank p-value of 0.033 compared to the tumors with CDC73 WT. GEP analysis revealed that the CDC73-mutated UCEC tumors had significant upregulation of immunological markers and enriched immunologic signature compared to the CDC73-WT tumors. In silico analysis of tumor immune microenvironment showed higher fractions of cytotoxic CD8 cells, T follicular helper cells and M1 macrophage in the CDC73-mutated UCEC tumors. The majority of the CDC73-mutated tumors were POLE ultra-mutated and microsatellite instability (MSI) subtypes, which likely account for a better survival and an increased immune-cell infiltrate of these tumors. Conclusion: CDC73 mutations is rare in general population and tumors except for parathyroid carcinoma and UCEC. CDC73 mutation is a marker for better prognosis in UCEC and is associated with increased immune cell infiltrate in tumor microenvironment, likely due to majority of the CDC73-mutated tumors were POLE ultra-mutated and microsatellite instability (MSI) subtypes.