Number Of CD8-positive Lymphocytes Research Articles

Abstract 5219: Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers

Abstract Background: Loss of chromosome Y (LOY) occurs commonly in cancers of male patients. It was recently proposed to be associated with cancer aggressiveness and altered T-cell function in bladder cancer and it was found to be linked to poor prognosis in a TCGA bladder cancer patient cohort of 300 patients based on a combined analysis of RNAs from Y-chromosome genes. In this study, we aimed to clarify the clinical relevance of LOY in a large cohort of urothelial bladder cancer patients by using fluorescence in-situ hybridization (FISH). Design: X and Y chromosomes were analyzed by FISH on a tissue microarray (TMA) containing 1,819 urothelial carcinomas of the urinary bladder from male patients including 487 patients who had undergone cystectomy for muscle-invasive disease and for which follow-up data were available. Data of tumor microenvironment parameters were obtained in a previous study. Results: LOY was found in 26.0% of 1,479 analyzable cancers. In the subset of non-invasive cancers, the frequency of LOY increased from pTaG2 (14.5%) to pTG3 (28.6%) but these differences were not significant (p=0.1015). In muscle-invasive cancers, the frequency of LOY increased slightly from pT2 (25.4%) to pT4 cancers (32.8%) but this association was again not significant (p=0.1868). Within pT2-4 cancers, LOY was associated with venous invasion (p=0.0010) but unrelated to pT, pN, and L-status, as well as to overall, recurrence-free, and cancer-specific survival. Muscle-invasive urothelial carcinomas with and without LOY did not show significant differences in the number of CD8 positive lymphocytes, the fraction of CD8 positive intraepithelial lymphocytes, the number of macrophages and dendritic cells, as well as the fraction of t helper and t regulatory cells. Conclusion: FISH is the gold standard for determining LOY. That LOY was neither associated with histopathological parameters of cancer aggressiveness and patient prognosis nor with any parameters describing the tumor microenvironment strongly argues against a driving role of LOY for bladder cancer progression and cancer associated immune reaction. The slight increase of LOY from pTaG2 to pT4 cancers is consistent with a continuous (but clinically irrelevant) loss of the dispensable Y chromosomes during tumor evolution over time. Citation Format: Martina Kluth, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberg, Antonia Franz, Annika Fendler, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Niclas C. Blessin, Maximilian Lennartz, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Joachim Weischenfeldt, Tobias Klatte, Sarah Minner, David Horst, Thorsten Schlomm, Dr. med Henrik Zecha Zecha. Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers [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 5219.

Approaching the tumor microenvironment in patients with advanced hepatocellular carcinoma using needle biopsy samples.

334 Background: Currently, combined immunotherapy of atezolizumab (anti-PD-L1 antibody) plus bevacizumab (a humanized anti-VEGF monoclonal antibody) is the standard first-line treatment in patients with advanced hepatocellular carcinoma (HCC). At the threshold of this new era, there is limited information about tumor microenvironment (TME) in advanced HCC. Several studies on TME in HCC have analyzed samples obtained via hepatic resection. In general, hepatic resection is indicated for patients with limited size and number of intrahepatic nodules, i.e., early stage HCC. In contrast, most patients who have an indication for systemic therapy have developed macroscopic vascular invasion (MVI) or/and extrahepatic metastasis, namely in advanced stage HCC. Progression from an early stage HCC to an advanced stage HCC involves a lengthy clinical course, therefore, the TME at the time of initial diagnosis may differ from that at the time of systemic therapy indication. The present study was aimed to analyze the TME by using needle biopsy samples obtained prior to initiation of systemic therapy in patients with advanced HCC. Methods: Between March 2019 and May 2020, 80 patients underwent liver tumor biopsy at the time of indication for systemic chemotherapy. HCC was confirmed via pathological examination in 70 patients and their samples were analyzed. Microsatellite instability (MSI) was evaluated using polymerase chain reaction. Programed death-ligand 1 (PD-L1) expression and the levels of tumor-infiltrating lymphocytes (TIL) were evaluated using immunohistochemical staining. PD-L1 expression was defined as per the tumor proportion score (TPS; the number of PD-L1-positive cells/total number of tumor cells) and was classified as low (TPS < 1%) or high (TPS >1%). Levels of TIL were defined as the mean number of CD8-positive lymphocytes in the tumor per 1 mm2 and classified as low or high using the median value. Results: Out of the 70 tumors, one was MSI-high and 69 were MSI-negative. The PD-L1 expression was < 1% in 50 samples, 1%–10% in 12, 11%–20% in 7, and 21%–30% in 1. The median level of TIL was 266/mm2. PD-L1highTILhighwas present in 20.0%, PD-L1lowTILlowin 38.5%, PD-L1highTILlowin 8.6%, and PD-L1lowTILhighin 32.9%. In the MSI-high tumor, PD-L1 expression was < 1% and the level of TIL was 142/mm2. High PD-L1 expression and high levels of TIL were associated with hepatitis C virus infection, high alpha-fetoprotein levels, and presence of MVI respectively. We are currently performing RNA-sequencing in order to obtain more details about TME in patients with advanced HCC. Conclusions: MSI-high advanced HCC was detected in 1.4% patients and was not necessarily associated with a “hot” immune microenvironment. PD-L1 expression and levels of TIL were associated with some clinical parameters. In the present study, we also reported the changes in the TME over time.

Abstract A40: Prevalence of TIGIT expression in normal tissues, inflammation, and cancer

Abstract Introduction: TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an immune checkpoint protein expressed on subsets of cytotoxic and regulatory T cells. TIGIT inhibiting drugs are currently being developed. The purpose of this study was to investigate the pattern of TIGIT expressing cells in normal tissues, inflammation and cancer. Methods: Monoclonal mouse antibodies were used for immunohistochemical TIGIT (Dianova, Hamburg, Germany) and PD1 (Abcam, Cambridge, UK, ab52587) analyses on formalin fixed paraffin embedded tissue sections from normal lymphatic tissues as well as selected inflammations and cancers. Results: In lymph nodes and tonsils, TIGIT positivity was seen in a large fraction of the germinal center T-cells. A high density of TIGIT positive T-cells also occurred in the marginal zone and in the interfollicular area. The analyses of selected inflammatory diseases revealed detectable TIGIT expression in a high proportion of T-lymphocytes in BCG induced cystitis and prostatitis, Hashimoto thyreoiditis, Lichen sclerosis of the penis, skin eczema, Crohn’s disease, and rheumatoid synovialitis. In all normal and inflammatory tissues, the pattern of TIGIT expression largely paralleled the pattern of PD1 expression. Three examples each of cancers types for which immune checkpoint inhibitors are already approved or extensively studied (melanoma, lung, kidney, bladder and colorectal cancer) were also analyzed. Here, the number of CD8 positive lymphocytes varied greatly between individual cases and the fraction of TIGIT and PD1 positive CD4 and CD8 positive lymphocytes varied considerably between cases. Conclusion: Expression of TIGIT is regularly seen in a large subset of T-cells and has a similar expression pattern as PD1. TIGIT’s frequent co-expression with PD1 in cytotoxic T-cells is consistent with TIGIT representing a clinically relevant druggable immune checkpoint regulator that potentially could be targeted in combination with PD1. Citation Format: Andrea Hinsch, Martina Kluth, Andreas M. Lübke, Anne Menz, Till Krech, Stefan Steurer, Doris Höflmayer, Guido Sauter, Waldemar Wilczak, Kristine Fischer, Ronald Simon. Prevalence of TIGIT expression in normal tissues, inflammation, and cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A40.

Evaluation of the effect and mechanism of action of local phenytoin in treatment of vitiligo

ABSTRACTThere are many theories explaining vitiligo such as genetic, autoimmune, neural, free radicals, biochemical, intrinsic defect, melanocytorrhagy, and convergent theories. Phenytoin is a widely used anticonvulsant, which is used in cutaneous medicine for treatment of ulcers and epidermolysis bullosa. The aim of this study is to assess the effectiveness of topical phenytoin gel in the treatment of vitiligo patients and explaining the underlying mechanism using immunohistochemistry for evaluation of HMB45, CD4, and CD8. Only 9 patients out of 28 experienced response to phenytoin in the form of dull, white color change and light brown color. Post-phenytoin treatment biopsies showed decreased density of inflammation, increased melanin and increased HMB45 positive cells together with an increased number of CD4 positive lymphocytes and decreased number of CD8 positive lymphocytes. These observations did not reach significant level (P > 0.05). A high percentage of CD4 positive lymphocytes was significantly associated with a long duration of vitiligo (p = 0.03) and segmental vitiligo type (p = 0.02). The current study applied phenytoin as 2% concentrated gel for 3 months, which is a relatively short duration without observed side effects throughout the period. These results indicate that topical phenytoin of low concentrations may have beneficial effects through immunomodulatory activity by affecting CD4 and CD8 counts and subsequently the ratio between them. Further studies are recommended to combine phenytoin with other antivitiligo agents as local corticosteroids or phototherapy to clarify if it could potentiate their effects.

Neutrophil-to-lymphocyte ratio is a prognostic marker in bladder cancer patients after radical cystectomy.

BackgroundThere is no reliable biomarker for predicting the prognosis of patients who undergo radical cystectomy for bladder cancer. Recent studies have shown that the neutrophil-to-lymphocyte ratio (NLR) could function as a useful prognostic factor in several types of malignancies. This study aimed to assess the usefulness of NLR in bladder cancer.MethodsA total of 74 patients who underwent radical cystectomy in our institutions from 1999 to 2014 were analyzed. The NLR was calculated using the patients’ neutrophil and lymphocyte counts before radical cystectomy. An immunohistochemical analysis was also performed to detect tumor infiltrating neutrophils (CD66b) and lymphocytes (CD8) in bladder cancer specimens.ResultsA univariate analysis showed that the patients with a high NLR (≥2.38; HR = 4.84; p = 0.007), high C-reactive protein level (>0.08; HR = 10.06; p = 0.030), or pathological lymph node metastasis (HR = 4.73; p = 0.030) had a significantly higher risk of cancer-specific mortality. Kaplan-Meier and log-rank tests further revealed that NLR was strongly correlated with overall survival (p = 0.018), but not progression-free survival (p = 0.137). In a multivariate analysis, all of these were found to be independent risk factors (HR = 4.62, 10.8, and 12.35, respectively). The number of CD8-positive lymphocytes was significantly increased in high-grade (p = 0.001) and muscle-invasive (p = 0.012) tumors, in comparison to low-grade and non-muscle-invasive tumors, respectively.ConclusionsThe NLR predicted the prognosis of patients who underwent radical cystectomy and might therefore function as a reliable biomarker in cases of invasive bladder cancer.

Associations of HIV testing and late diagnosis at a Japanese university hospital

OBJECTIVES:This study was conducted to clarify the rate of late diagnosis of HIV infection and to identify relationships between the reasons for HIV testing and a late diagnosis.METHODS:This retrospective cohort study was conducted among HIV-positive patients at the Jikei University Hospital between 2001 and 2014. Patient characteristics from medical records, including age, sex, sexuality, the reason for HIV testing and the number of CD4-positive lymphocytes at HIV diagnosis, were assessed.RESULTS:A total of 459 patients (men, n=437; 95.2%) were included in this study and the median age at HIV diagnosis was 36 years (range, 18–71 years). Late (CD4 cell count <350/mm3) and very late (CD4 cell count <200/mm3) diagnoses were observed in 61.4% (282/459) and 36.6% (168/459) of patients, respectively. The most common reason for HIV diagnosis was voluntary testing (38.6%, 177/459 patients), followed by AIDS-defining illness (18.3%, 84/459 patients). Multivariate analysis revealed a significant association of voluntary HIV testing with non-late and non-very-late diagnoses and there was a high proportion of AIDS-defining illness in the late and very late diagnosis groups compared with other groups. Men who have sex with men was a relative factor for non-late diagnosis, whereas nonspecific abnormal blood test results, such as hypergammaglobulinemia and thrombocytopenia, were risk factors for very late diagnosis.CONCLUSIONS:Voluntary HIV testing should be encouraged and physicians should screen all patients who have symptoms or signs and particularly hypergammaglobulinemia and thrombocytopenia, that may nonspecifically indicate HIV infection.

Programmed Death 1 Ligand (PDL-1) Expression in Colorectal Cancer (CRC) Correlates With Lymph Node Metastasis

Introduction: Expression of PDL-1 in tumor cells is one of the mechanisms by which tumors suppress immune surveillance, enabling them to invade and metastasize. Antibodies targeting PD1 and PDL-1 are currently being used in clinical trials and showed significant benefit in a variety of tumor types. In one large series of CRC, high PDL-1 expression was found to correlate inversely with lymph node metastasis, in contrast with what is reported in other solid tumors (Droeser et al 2013); in that study PDL-1 was scored manually using visual analog scale of immunohistochemically (IHC) stained sections. The aim of this study was to investigate the relationship between PDL-1 expression in CRC with nodal status using quantitative image analysis. Methods: Sections of 70 samples of CRC were stained by IHC using specific antibodies to PDL-1 and CD8. The level of PDL-1 expression was determined using the image analysis software OTMIAS and associated PDL-1 standard. The number of CD8 positive lymphocytes was determined by direct counting using a grid and a cell counter. Statistical analysis was performed using Prism statistical analysis software. Results: CRC with high levels of PDL-1 expression (>70% of tumor cells with > 20 units of PDL-1 per cell) were more likely to be associated with lymph node metastasis (7 of 13 or 54%) than CRC with lower PDL-1 expression (14 of 57 or 25%) (p < 0.05). There was no significant association between PDL-1 expression and depth of tumor invasion. The mean number of CD8 positive lymphocytes in CRC with high PDL-1 expression was less (18) than that with lower PDL-1 (24) although the difference did not reach statistical significant (p=0.06). Conclusion: Our finding, using quantitative image analysis, show that high PDL-1 levels in CRC are associated with increased incidence of lymph node metastasis, similar to what have been reported for other solid tumors. Therefore, patients with CRC expressing high levels of PDL-1 may benefit from related targeted therapy.

IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer

Background:PD-L1 (programmed cell death 1 ligand 1) on tumour cells suppresses host immunity through binding to its receptor PD-1 on lymphocytes, and promotes peritoneal dissemination in mouse models of ovarian cancer. However, how PD-L1 expression is regulated in ovarian cancer microenvironment remains unclear.Methods:The number of CD8-positive lymphocytes and PD-L1 expression in tumour cells was assessed in ovarian cancer clinical samples. PD-L1 expression and tumour progression in mouse models under conditions of altering IFN-γ signals was assessed.Results:The number of CD8-positive cells in cancer stroma was very high in peritoneally disseminated tumours, and was strongly correlated to PD-L1 expression on the tumour cells (P<0.001). In mouse models, depleting IFNGR1 (interferon-γ receptor 1) resulted in lower level of PD-L1 expression in tumour cells, increased the number of tumour-infiltrating CD8-positive lymphocytes, inhibition of peritoneal disseminated tumour growth and longer survival (P=0.02). The injection of IFN-γ into subcutaneous tumours induced PD-L1 expression and promoted tumour growth, and PD-L1 depletion completely abrogated tumour growth caused by IFN-γ injection (P=0.01).Conclusions:Interferon-γ secreted by CD8-positive lymphocytes upregulates PD-L1 on ovarian cancer cells and promotes tumour growth. The lymphocyte infiltration and the IFN-γ status may be the key to effective anti-PD-1 or anti-PD-L1 therapy in ovarian cancer.

Su1879 Lymphocytic Esophagitis With or With No Apoptoses: Two Unequal Subgroups in the Wide Ranging Concept of Lymphocytic Esophagitis

BACKGROUND& AIMS: Lymphocytic is ill defined and inflammatory lesions of esophageal squamous epithelium are believed to show non-specific histological changes. This study was performed to investigate the histological and molecular criteria in esophagitis. METHODS: We collected 33 biopsies from 28 patients with esophagitis, which were diagnosed between 2010 and 2012 (17 men and 11 women, mean age = 60,8 and 61,1 years, respectively). Grading was performed by the number of lymphocytes (50/HPF cut off value) and according to Purdy et al. The inflammatory infiltrate was characterized by using the XTRACT robot (STRATIFYER, Cologne, Germany), quantitative real-time-PCR (FOXP3, CXCL9, CXCL13, IGHM, CD3, CD4 and CD8) and immunohistochemistry (CD4, CD8, TIA, GranzymB and Caspase 3). Spearman rank test was used to calculate the correlation between these parameters. RESULTS: Apoptoses could be detected in 83% of severe cases (15/18) and in 47% of mild cases (7/15) without differences between the two grading systems. High levels of cytotoxic T-cells were present in 80% of severe cases with apoptoses (12/15) and in 5 cases without visible apoptoses in H&E stain, mostly mild cases. The overall number of lymphocytes correlated with the number of apoptoses (p = 0,002) and the number of CD8 positive lymphocytes (p = 0,005). Cases with apoptoses showed high levels of CD8 positive lymphocytes. A positive correlation between spongiosis and levels of cytotoxic T-cells could be demonstrated (p = ,0,0001). Evaluation of clinical data revealed a correlation between high levels of cytotoxic lymphocytes and a wavey mucosal surface (p = 0,025), reddening of esophageal epithelium (p = 0,017) and a stipplelike exsudate (p = 0,015). Within the group with apoptoses a strong correlation between cytotoxic T-cells (= CD8) and regulatory T-cells (= FOXP3) (p = ,0,0001) was found. Expression of CXCL9 correlated with the expression of CD4 and CD8 (p = 0,05 and p = 0,0042, respectively). CONCLUSIONS: Our data suggest that the term lymphocytic esophagitis should only be used as an umbrella-term in several diseases and therefore we suggest lymphocyte-rich esophagitis to be used for further subdivisions. Two different subgroups of this variant of chronic can be distinguished: Lymphocyte-rich with apoptosis (LEA) and with no apoptosis (LENA). Apoptotic keratinocytes are usefull in grading LEA, as they can be found in large numbers predominantely in severe forms. The number and localization of apoptoses distinguishes esophageal Crohn's disease and lichen planus from LEA. Further studies should focus on the question, whether our findings are a hint to an autoimmunologic mechanism or not.

Ultrastructural types of alveolar macrophages in bronchoalveolar lavages from patients with pulmonary sarcoidosis

By routine applied quantitative BAL methods are particularly helpful for the diagnosis of pulmonary sarcoidosis. Here the morphology of the alveolar cells does not play a role. However, morphological and especially electron microscopic investigations might contribute to the clarification of the aetiology of this disease. In a prospective study we investigated the bronchoalveolar lavages (BALs) from 10 patients with recently histologically diagnosed, untreated pulmonary sarcoidosis. Commonly applied cytological and immunological BAL diagnostic techniques were accompanied by morphological investigations of alveolar cells, especially alveolar macrophages, using light and electron microscopy. All patients showed lymphocytic alveolitis with an increased number of CD4 positive lymphocytes as well as an increased CD4/CD8 ratio. A striking light microscopic finding was the great morphological variety of the alveolar macrophages. Electron microscopy revealed typical lymphocytes, neutrophils, and eosinophils as well as three different types of alveolar macrophages in all 10 patients: type I (approx. 30%) with a normal macrophage morphology, a vacuole-rich type II (approx. 30%) with myelin-like structures and type III (approx. 40%) with electron-dense inclusions. The occurrence of intracellular myelin figures in type II macrophages is a hint for increased phagocytotic processes of surfactant with or without its overproduction in the sense of a secondary alveolar proteinosis. Numerous electron-dense inclusions in type III also indicate an increased macrophage activity that leads to an increased release of cytokines, which in turn can trigger an inflammatory reaction.

Efficacy and Safety of a Monoclonal Purified Factor VIII Concentrate: 5-Year Follow-Up in Previously Treated HIV-Negative Haemophiliacs

The efficacy and safety of a monoclonal purified factor VIII concentrate (Hemofil M) were assessed in a historically controlled study in 22 HIV-negative patients with haemophilia A, previously treated with various concentrates. Data from 5 years of follow-up were compared with those from the preceding 6 months. Factor VIII consumption remained unchanged. A temporary increase in the number of reported bleedings was attributed to more frequent follow-up visits in the first year. Allergic reactions, inhibitor formation and HIV infection were not seen. Liver function parameters fluctuated in individual patients, and were not related to the ultrapure concentrate used. No clinical evidence of liver insufficiency was seen. The number of CD4-positive lymphocytes was stable, while platelet numbers showed a remarkable increase. We conclude that in previously treated HIV-negative haemophiliacs, treatment with a monoclonal purified factor VIII concentrate is efficacious and safe with regard to HIV transmission, allergic reactions, induction of inhibitors, and deterioration of immune abnormalities.

Alterations in spliced and unspliced HIV-1-specific RNA detection in peripheral blood mononuclear cells of individuals with varying CD4-positive lymphocyte counts.

Reverse transcriptase-polymerase chain amplification reactions (RT-PCR) were used to identify transcripts for HIV-1 structural and regulatory proteins in peripheral blood mononuclear cells of a cohort of 48 patients. At least one set of PCR primers was capable of detecting HIV-1 transcripts in 94% of patients. Unspliced gag-pol transcripts were detected with gag or pol primer sets in 60 and 63% of samples, respectively. A significant inverse correlation was noted between transcript identification with the gag primer set and the number of CD4-positive lymphocytes in the blood sample and the clinical stage of infection. Single-spliced env transcripts were identified in 44% of individuals. Multiple-spliced tat or nef transcripts were detected in 6.2 and 53% of individuals, respectively. These findings indicate that viral transcripts are expressed throughout the course of HIV-1 infection.