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Immunotherapy is a new anti-cancer treatment option, showing promising results in clinical trials. To investigate potential immune biomarkers in esophageal adenocarcinoma (EAC), we explored immune landscape patterns in the tumor microenvironment before and after neoadjuvant chemoradiation (nCRT). Sections from matched pretreatment biopsies and post-nCRT resection specimens (n = 188) were stained for (1) programmed death-ligand 1 (PD-L1, CD274); (2) programmed cell death protein 1 (PD-1, CD279), forkhead box P3 (FOXP3), CD8, pan-cytokeratin multiplex; and (3) an MHC class I, II duplex. The densities of tumor-associated immune cells (TAICs) were calculated using digital image analyses and correlated to histopathological nCRT response [tumor regression grade (TRG)], survival, and post-nCRT immune patterns. PD-L1 positivity defined by a combined positive score of >1 was associated with a better response post-nCRT (TRG 1-3 versus 4, 5, p = 0.010). In addition, high combined mean densities of CD8 + , FOXP3 + , and PD-1 + TAICs in the tumor epithelium and stroma of biopsies were associated with a better response (TRG 1-3 versus 4, 5, p = 0.025 and p = 0.044, respectively). Heterogeneous TAIC density patterns were observed post-nCRT, with significantly higher CD8 + and PD-1 + TAIC mean densities compared with biopsies (both p = 0.000). Three immune landscape patterns were defined post-nCRT: 'inflamed', 'invasive margin', and 'desert', of which 'inflamed' was the most frequent (57%). Compared with matched biopsies, resection specimens with 'inflamed' tumors showed a significantly higher increase in CD8 + density compared with non-inflamed tumors post-nCRT (p = 0.000). In this cohort of EAC patients, higher TAIC densities in pretreatment biopsies were associated with response to nCRT. This warrants future research into the potential of the tumor-immune landscape for patient stratification and novel (immune) therapeutic strategies. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Clinical outcome in patients with diffuse large B cell lymphomas (DLBCL) is poorly predictable. Expression of proteins related to germinal centre B (GCB) cell or activated B cells (ABC) and expression of apoptosis-regulating proteins Bcl-2 and XIAP have been found previously to be strongly associated with clinical outcome. In this study we aimed to develop an algorithm based on expression of GCB/ABC-related proteins CD10, Bcl-6 and MUM1 and apoptosis-inhibiting proteins Bcl-2, XIAP and cFLIP for optimal stratification of DLBCL patients into prognostically favourable and unfavourable groups. Expression of CD10 and cFLIP was associated with better overall survival (both p = 0.03), whereas expression of MUM1, Bcl-2 and XIAP was associated with poor clinical outcome (p = 0.01, p = 0.0007 and p = 0.03, respectively). Multivariate analysis revealed that Bcl-2 was the strongest prognostic marker followed by CD10 and MUM1. Stratification of patients according to a new algorithm based on expression of these three markers improved patient risk stratification into low and particularly high clinical risk groups (p = 0.04 and p < 0.0001, respectively). We conclude that, in our group of primary nodal DLBCLs, a new algorithm, based on expression of the apoptosis-inhibiting protein Bcl-2 and the GCB/ABC-related proteins CD10 and MUM1, strongly predicts outcome in International Prognostic Index (IPI)-low and -high patients. Its predictive power is stronger than previously published algorithms based on only GCB/ABC- or apoptosis-regulating proteins.

This study determined whether tumour-infiltrating lymphocytes (TILs) in nasopharyngeal carcinomas (NPCs) include activated cytotoxic T lymphocytes (CTLs) and whether the numbers of activated CTLs in these biopsies are related to clinical outcome. Moreover, the study investigated whether the numbers of activated CTLs are associated with the expression of MHC class I proteins and the granzyme B antagonist PI-9 in the tumour cells. Forty-three Indonesian NPC patients (T(1-3), N(1-3), M(0)), who were treated with curative intent by radiotherapy only, were studied. Tumour-infiltrating activated CTLs were detected using antibodies against granzyme B, CD8, and CD56. Expression of MHC class I proteins and PI-9 was also determined by immunohistochemistry. Granzyme B-positive TILs were detected in all NPC biopsies. The presence of a high percentage (>25%) of granzyme B-positive TILs appeared to be a very strong predictor of a rapid fatal clinical outcome, independent of stage. Complete absence of MHC class I heavy chain expression in tumour cells was observed in 11 of 31 evaluable cases and low levels were observed in seven additional cases. No association between MHC class I expression and the numbers of granzyme B-positive TILs was observed. Expression of the granzyme B antagonist PI-9 in tumour cells was detected in three cases. It is concluded that the presence of many granzyme B-positive TILs in a selected group of Indonesian NPC patients is a strong and stage-independent marker for a rapid fatal clinical outcome., (Copyright 2002 John Wiley & Sons, Ltd.)

In vitro studies indicate that in lymphomas, execution of apoptosis involves activation of effector caspases. To investigate activation of effector caspases in vivo in biopsy specimens of lymphomas, a new assay was developed using antibodies against active caspase 3 and p89, a protein fragment generated by caspase-specific cleavage of poly-ADP ribose polymerase (PARP). Using this assay, it was found that in B-cell lymphomas, levels of active caspase 3/p89-positive cells correlate strongly with morphologically recognizable apoptotic cells. The number of active caspase 3/p89-positive cells was low in follicular lymphomas and usually high in diffuse large cell lymphomas. Highest numbers were found in Burkitt lymphomas and in two biopsies of diffuse large B-cell lymphomas (DLCLs) obtained several days after initiation of therapy. It is concluded that apoptosis in reactive lymphoid tissues and in B-cell lymphomas always involves activation of effector caspase 3 and cleavage of one of the major effector caspase substrates, PARP-1. Moreover, levels of effector caspase activation are constantly low in low-grade follicular lymphomas and vary considerably in DLCL and Burkitt lymphoma., (Copyright 2002 John Wiley & Sons, Ltd.)

Hodgkin's disease (HD) is a malignant lymphoproliferative disease characterized by the presence of Hodgkin-Reed-Sternberg cells surrounded by a reactive infiltrate. In Epstein-Barr virus (EBV)-associated cases (40-60%), at least two EBV-encoded proteins [latent membrane protein 1 (LMP1) and LMP2] are expressed, which are potential targets for cytotoxic T-lymphocytes (CTLs). Although in EBV-positive cases significantly more activated (granzyme B-positive) CTLs and natural killer (NK) cells are present, the cytotoxic immune response is not sufficient for adequate killing of tumour cells. The production of immunomodulating cytokines within the tumour may be one of the mechanisms causing circumvention of the immune system. This study investigated by immunohistochemistry the presence of the immunosuppressive cytokine interleukin-10 (IL-10) and other Th1/Th2-associated cytokines [IL-2, IL-4, interferon-gamma (IFN-gamma)] in the neoplastic cells and reactive lymphocytes of nine EBV-positive and 18 EBV-negative cases of HD. The percentage of IL-10-expressing cells, both neoplastic and reactive, in EBV-positive cases was significantly higher (33.1% vs. 18.5% for the neoplastic cells and 21.6% and 12.2% for the reactive cells, p=0.003 and 0.04, respectively) than in EBV-negative cases. No difference in the percentage of IL-2-, IL-4- and IFN-gamma-expressing cells was observed. These results suggest that escape from local immune surveillance is not due to a shift from Th1 towards Th2, but may be caused by a direct effect of IL-10 on the cytotoxic cells., (Copyright 2000 John Wiley & Sons, Ltd.)

Epstein-Barr virus (EBV) has been demonstrated in the Reed-Sternberg cells and their mononuclear variants (Hodgkin cells; H-RS cells) in a substantial number of Hodgkin's disease (HD) cases. Moreover, EBV can modulate both in vivo and in vitro the expression of several cellular genes, including lymphoid differentiation markers. Therefore we investigated, in 64 cases of HD, the relationship between the presence of EBV and the expression of lymphoid (CD45RB), T- (CD3, CD45RO), B- (CD20, MB2 antigen, CDw75), and myeloid-cell lineage markers (CD15), and of activation markers (CD30, EMA, and the 115D8 antigen) on the H-RS cells. EBV-positive cases, as demonstrated by the presence of EBER-1 and -2 RNA and LMP-1 protein expression, showed a significant reduction in the expression on H-RS cells of T-cell lineage (CD3, P < 0.02), B-cell lineage (CD20; P < 0.005), and activation markers (EMA; P < 0.002 and the 115D8 antigen; P < 0.001) as compared with EBV-negative cases. No differences were found in the expression of CD15, CD30, CD45RO, CD45RB, CDw75, or the MB2 antigen on H-RS cells in EBV-positive and EBV-negative HD cases. Interestingly, in 11 cases of EBV-negative HD, B- as well as T-cell lineage markers could be found on some H-RS cells. These data suggest that EBV in H-RS cells is able to down-regulate the expression of T- (CD3) and B- (CD20) cell lineage markers and lymphoid activation markers (EMA and the 115D8 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)

Forty-four cases of Hodgkin's disease (HD), mostly of the nodular sclerosing type, were investigated for the presence of Epstein-Barr virus (EBV) by polymerase chain reaction (PCR) and DNA and RNA in situ hybridization (DISH, RISH), as well as by immunohistochemistry for the detection of latent membrane protein-1 (LMP-1) of EBV. In situ hybridization (ISH) was combined with immunohistochemistry to correlate the presence and activity of the virus at the cellular level. In 18/34 (53 per cent) cases, EBV-DNA sequences could be detected with the PCR method. In 12/18 positive cases, DISH and RISH were also positive. In the remaining six EBV-PCR positive cases, two were also positive with RISH and LMP-1, whereas no positive signal with DISH could be obtained. All DISH and/or RISH positive cases were also positive for LMP-1. With RISH, not only the Reed-Sternberg cells and their mononuclear variants (RS cells) stained positive, but also small and intermediate cells frequently reacted with the EBV-specific probes (EBER-1 and -2). Double staining with cellular markers (CD3, CD20, CD45, CD45RO, CD68, and the lectin PNA) revealed that most of the smaller EBER-positive cells frequently did not express T, B, or histiocytic markers, but that they, as well as the RS cells, showed cytoplasmic and membranous staining with PNA. These smaller EBER-positive cells were not found in EBV-PCR negative HD. EBER-positive RS cells were almost always LMP-1 positive, as well as a substantial proportion of the intermediate-sized cells, whereas the majority of the small EBER-positive cells remained LMP-1 negative.(ABSTRACT TRUNCATED AT 250 WORDS)