Publications by authors named "Joe Poh Sheng Yeong"

20 Publications

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Pan-cancer analysis of ligand-receptor crosstalk in the tumor microenvironment.

Cancer Res 2021 Feb 5. Epub 2021 Feb 5.

Computational and Systems Biology, Genome Institute of Singapore

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is key to tumor progression. Here we deconvoluted bulk tumor transcriptomes to infer crosstalk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. This approach recovered known transcriptional hallmarks of cancer and stromal cells and was concordant with single-cell, in-situ hybridization and immunohistochemistry data. Inferred autocrine cancer cell interactions varied between tissues but often converged on Ephrin, BMP, and FGFR-signaling pathways. Analysis of immune checkpoints nominated interactions with high levels of cancer-to-immune crosstalk across distinct tumor types. Strikingly, PD-L1 was found to be highly expressed in stromal rather than cancer cells. Overall, our study presents a new resource for hypothesis generation and exploration of crosstalk in the TME.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2352DOI Listing
February 2021

Delineating the breast cancer immune microenvironment in the era of multiplex immunohistochemistry/immunofluorescence (mIHC/IF).

Histopathology 2021 Jan 5. Epub 2021 Jan 5.

Integrative Biology for Theranostics, Institute of Molecular Cell Biology, Agency of Science, Technology and Research (A*STAR), Singapore, 138673, Singapore.

Breast cancer is the commonest malignancy and the leading cause of cancer death in females worldwide (1). Treatment is challenging especially for those that are triple negative. Increasing evidence suggests that diverse immune populations are present in the breast tumour microenvironment, which open up avenues for personalised drug targets. Historically, our investigations into the immune constitution of breast tumours have been restricted to analyses of one or two markers at a given time. Recent technological advances have allowed simultaneous labelling of over 35 markers, detailed profiling of tumour immune infiltrates at the single-cell level, as well as determining the cellular composition and spatial analysis of the entire tumour architecture. In this review, we describe emerging technologies that have contributed to the field of breast cancer diagnosis, and discuss how to interpret the vast datasets obtained in order to effectively translate them for clinically relevant use.
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http://dx.doi.org/10.1111/his.14328DOI Listing
January 2021

Partial absence of PD-1 expression by tumor-infiltrating EBV-specific CD8 T cells in EBV-driven lymphoepithelioma-like carcinoma.

Clin Transl Immunology 2020 9;9(9):e1175. Epub 2020 Sep 9.

Vaccine and Infectious Disease Division Fred Hutchinson Cancer Research Center Seattle WA USA.

Objectives: Lymphoepithelioma-like carcinoma (LELC) is an uncommon lung cancer, typically observed in young, non-smoking Asian populations. LELC is associated with Epstein-Barr virus (EBV) infection of lung tumor cells of epithelial origin, suggesting a carcinogenic role of EBV as observed in nasopharyngeal carcinoma (NPC). Here, we studied the antigen specificity and phenotype of EBV-specific CD8 T cells in blood and tumor of one LELC patient positive for EBV infection in lung tumor cells.

Methods: Using multiplex MHC class I tetramers, mass cytometry and mRNA sequencing, we studied EBV-specific CD8 T cells at the transcriptomic and phenotypic levels in blood and tumor tissues of the LELC patient.

Results: Lymphoepithelioma-like carcinoma lung tumor cells were positive for EBV infection. In both blood and tumor tissues, we detected two populations of EBV-specific CD8 T cells targeting the EBV lytic cycle proteins: BRLF1 and BMLF1. Transcriptomic analyses of these two populations in the tumor, which can be considered as tumor-specific, revealed their distinct exhausted profile and polyclonal TCR repertoire. High-dimensional phenotypical analysis revealed the distinct phenotype of these cells between blood and tumor tissues. In tumor tissue, EBV-specific CD8 TILs were phenotypically heterogeneous, but consistently expressed CD39. Unexpectedly, although the LELC tumor cells expressed abundant PD-L1, these tumor-specific CD8 tumor-infiltrating lymphocytes (TILs) mostly did not express PD-1.

Conclusion: Epstein-Barr virus-specific CD8 TILs in EBV-driven tumor are heterogeneous and partially lack PD-1 expression, suggesting that anti-PD1/PD-L1 immunotherapy may not be an appropriate strategy for disinhibiting EBV-specific cells in the treatment of LELC patients.
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http://dx.doi.org/10.1002/cti2.1175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503213PMC
September 2020

Transcriptional Spatial Profiling of Cancer Tissues in the Era of Immunotherapy: The Potential and Promise.

Cancers (Basel) 2020 Sep 9;12(9). Epub 2020 Sep 9.

Institute of Molecular and Cell Biology (IMCB), Agency of Science, Technology and Research (A*STAR), Singapore 169856, Singapore.

Intratumoral heterogeneity poses a major challenge to making an accurate diagnosis and establishing personalized treatment strategies for cancer patients. Moreover, this heterogeneity might underlie treatment resistance, disease progression, and cancer relapse. For example, while immunotherapies can confer a high success rate, selective pressures coupled with dynamic evolution within a tumour can drive the emergence of drug-resistant clones that allow tumours to persist in certain patients. To improve immunotherapy efficacy, researchers have used transcriptional spatial profiling techniques to identify and subsequently block the source of tumour heterogeneity. In this review, we describe and assess the different technologies available for such profiling within a cancer tissue. We first outline two well-known approaches, in situ hybridization and digital spatial profiling. Then, we highlight the features of an emerging technology known as Visium Spatial Gene Expression Solution. Visium generates quantitative gene expression data and maps them to the tissue architecture. By retaining spatial information, we are well positioned to identify novel biomarkers and perform computational analyses that might inform on novel combinatorial immunotherapies.
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http://dx.doi.org/10.3390/cancers12092572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563386PMC
September 2020

The Virological, Immunological, and Imaging Approaches for COVID-19 Diagnosis and Research.

SLAS Technol 2020 12 18;25(6):522-544. Epub 2020 Aug 18.

Institute of Molecular Cell Biology (IMCB), Agency of Science, Technology and Research (A*STAR), Singapore, Singapore.

In 2019, a novel coronavirus (SARS-CoV-2) was found to cause a highly contagious disease characterized by pneumonia. The disease (COVID-19) quickly spread around the globe, escalating to a global pandemic. In this review, we discuss the virological, immunological, and imaging approaches harnessed for COVID-19 diagnosis and research. COVID-19 shares many clinical characteristics with other respiratory illnesses.Accurate and early detection of the infection is pivotal to controlling the outbreak, as this enables case identification, isolation, and contact tracing. We summarize the available literature on current laboratory and point-of-care diagnostics, highlight their strengths and limitations, and describe the emerging diagnostic approaches on the horizon.We also discuss the various research techniques that are being used to evaluate host immunity in laboratory-confirmed patients. Additionally, pathological imaging of tissue samples from affected patients has a critical role in guiding investigations on this disease. Conventional techniques, such as immunohistochemistry and immunofluorescence, have been frequently used to characterize the immune microenvironment in COVID-19. We also outline the emerging imaging techniques, such as the RNAscope, which might also aid in our understanding of the significance of COVID-19-specific biomarkers, such as the angiotensin-converting enzyme 2 (ACE2) cellular receptor.Overall, great progress has been made in COVID-19 research in a short period. Extensive, global collation of our current knowledge of SARS-CoV-2 will provide insights into novel treatment modalities, such as monoclonal antibodies, and support the development of a SARS-CoV-2 vaccine.
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http://dx.doi.org/10.1177/2472630320950248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435207PMC
December 2020

IRF-7 Mediates Type I IFN Responses in Endotoxin-Challenged Mice.

Front Immunol 2020 16;11:640. Epub 2020 Apr 16.

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore.

IRF-7 mediates robust production of type I IFN via MyD88 of the TLR9 pathway in plasmacytoid dendritic cells (pDCs). Previous studies using bone marrow-derived dendritic cells lacking either or have demonstrated that only IRF-3 is required for IFN-β production in the TLR4 pathway. Here, we show that IRF-7 is essential for both type I IFN induction and IL-1β responses via TLR4 in mice. Mice lacking were defective in production of both IFN-β and IL-1β, an IFN-β-induced pro-inflammatory cytokine, after LPS challenge. IFN-β production in response to LPS was impaired in IRF-7-deficient macrophages, but not dendritic cells. Unlike pDCs, IRF-7 is activated by the TRIF-, but not MyD88-, dependent pathway via TBK-1 in macrophages after LPS stimulation. Like pDCs, resting macrophages constitutively expressed IRF-7 protein. This basal IRF-7 protein was completely abolished in either or macrophages, which corresponded with the loss of LPS-stimulated IFN-β induction in these macrophages. These findings demonstrate that macrophage IRF-7 is critical for LPS-induced type I IFN responses, which in turn facilitate IL-1β production in mice.
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http://dx.doi.org/10.3389/fimmu.2020.00640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176903PMC
April 2020

Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy.

Cancers (Basel) 2020 Apr 22;12(4). Epub 2020 Apr 22.

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673, Singapore.

Immune checkpoint blockade (ICB) monotherapy shows early promise for the treatment of nasopharyngeal carcinoma (NPC) in patients. Nevertheless, limited representative NPC models hamper preclinical studies to evaluate the efficacy of novel ICB and combination regimens. In the present study, we engrafted NPC biopsies in non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice and established humanized mouse NPC-patient-derived xenograft (NPC-PDX) model successfully. Epstein-Barr virus was detected in the NPC in both NSG and humanized mice as revealed by Epstein-Barr virus-encoded small RNA (EBER) in situ hybridization (ISH) and immunohistochemical (IHC) staining. In the NPC-bearing humanized mice, the percentage of tumor-infiltrating CD8 cytotoxic T cells was lowered, and the T cells expressed higher levels of various inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) than those in blood. The mice were then treated with nivolumab and ipilimumab, and the anti-tumor efficacy of combination immunotherapy was examined. In line with paired clinical data, the NPC-PDX did not respond to the treatment in terms of tumor burden, whilst an immunomodulatory response was elicited in the humanized mice. From our results, human proinflammatory cytokines, such as interferon-gamma (IFN-γ) and interleukin-6 (IL-6) were significantly upregulated in plasma. After treatment, there was a decrease in CD4/CD8 ratio in the NPC-PDX, which also simulated the modulation of intratumoral CD4/CD8 profile from the corresponding donor. In addition, tumor-infiltrating T cells were re-activated and secreted more IFN-γ towards ex vivo stimulation, suggesting that other factors, including soluble mediators and metabolic milieu in tumor microenvironment may counteract the effect of ICB treatment and contribute to the tumor progression in the mice. Taken together, we have established and characterized a novel humanized mouse NPC-PDX model, which plausibly serves as a robust platform to test for the efficacy of immunotherapy and may predict clinical outcomes in NPC patients.
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http://dx.doi.org/10.3390/cancers12041025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225949PMC
April 2020

Counting Mitoses With Digital Pathology in Breast Phyllodes Tumors.

Arch Pathol Lab Med 2020 11;144(11):1397-1400

From the Department of Anatomical Pathology (Chow, Thike, Tan, Nasir), Singapore General Hospital, Singapore.

Context.—: Mitotic count is an important histologic criterion for grading and prognostication in phyllodes tumors (PTs). Counting mitoses is a routine practice for pathologists evaluating neoplasms, but different microscopes, variable field selection, and areas have led to possible misclassification.

Objective.—: To determine whether 10 high-power fields (HPFs) or whole slide mitotic counts correlated better with PT clinicopathologic parameters using digital pathology (DP). We also aimed to find out whether this study might serve as a basis for an artificial intelligence (AI) protocol to count mitosis.

Design.—: Representative slides were chosen from 93 cases of PTs diagnosed between 2014 and 2015. The slides were scanned and viewed with DP. Mitotic counting was conducted on the whole slide image, before choosing 10 HPFs and demarcating the tumor area in DP. Values of mitoses per millimeter squared were used to compare results between 10 HPFs and the whole slide. Correlations with clinicopathologic parameters were conducted.

Results.—: Both whole slide counting of mitoses and 10 HPFs had similar statistically significant correlation coefficients with grade, stromal atypia, and stromal hypercellularity. Neither whole slide mitotic counts nor mitoses per 10 HPFs showed statistically significant correlations with patient age and tumor size.

Conclusions.—: Accurate mitosis counting in breast PTs is important for grading. Exploring machine learning on digital whole slides may influence approaches to training, testing, and validation of a future AI algorithm.
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http://dx.doi.org/10.5858/arpa.2019-0435-OADOI Listing
November 2020

Liver fibrosis and CD206 macrophage accumulation are suppressed by anti-GM-CSF therapy.

JHEP Rep 2020 Feb 6;2(1):100062. Epub 2019 Dec 6.

Program in Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore.

Background & Aims: Chronic liver inflammation leads to fibrosis and cirrhosis and is associated with an accumulation of intrahepatic TNFα-secreting CD206 macrophages, which may participate in maintaining chronic liver disease in a GM-CSF-dependent manner. We aimed to elucidate the exact role of GM-CSF in the development and progression of chronic liver disease.

Methods: Liver immunohistochemistry and serum quantification were performed in patients with viral and non-viral-related liver disease to compare CD206 monocyte/macrophages, fibrosis and GM-CSF. This was followed by functional validations and in humanised mice.

Results: Using multiplex immunofluorescence and histo-cytometry, we show that highly fibrotic livers had a greater density of CD206 macrophages that produced more TNFα and GM-CSF in the non-tumour liver regions of patients with hepatocellular carcinoma (n = 47), independent of aetiology. In addition, the absolute number of CD206 macrophages strongly correlated with the absolute number of GM-CSF-producing macrophages. In non-HCC chronic HCV patients (n = 40), circulating GM-CSF levels were also increased in proportion to the degree of liver fibrosis and serum viral titres. We then demonstrated that monocytes converted to TNFα-producing CD206 macrophage-like cells in response to bacterial products (lipopolysaccharide) in a GM-CSF-dependent manner, confirming the normalisation of serum GM-CSF concentration following oral antibiotic treatment observed in HBV-infected humanised mice. Finally, anti-GM-CSF neutralising antibody treatment reduced intrahepatic CD206 macrophage accumulation and abolished liver fibrosis in HBV-infected humanised mice.

Conclusions: While the direct involvement of CD206 macrophages in liver fibrosis remains to be demonstrated, these findings show that GM-CSF may play a central role in liver fibrosis and could guide the development of anti-GM-CSF antibody-based therapy for the management of patients with chronic liver disease.

Lay Summary: Liver fibrosis is a major driver of liver disease progression. Herein, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the development of liver fibrosis. Our findings support the use of anti-GM-CSF neutralising antibodies for the management of patients with chronic liver disease resulting from both viral and non-viral causes.
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http://dx.doi.org/10.1016/j.jhepr.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005658PMC
February 2020

Tertiary lymphoid structures and associated plasma cells play an important role in the biology of triple-negative breast cancers.

Breast Cancer Res Treat 2020 Apr 7;180(2):369-377. Epub 2020 Feb 7.

Division of Pathology, Department of Anatomical Pathology, Singapore General Hospital, 20 College Road, Academia, Level 10, Singapore, 169856, Singapore.

Purpose: Triple-negative breast cancers (TNBC) are aggressive tumours that exhibit abundant lymphoid infiltrates which modulate tumour behaviour. Recent findings suggest that TNBC with higher densities of plasma cells are associated with a favourable prognosis, and tertiary lymphoid structures (TLS) have prognostic significance. Here, we studied the phenotype and function of plasma cells in TNBCs by assessing their association with IgG Kappa light chain expression, B cells, and TLS.

Methods: A retrospective analysis of 269 TNBC cases was performed. Tumour-infiltrating CD38+ plasma cells, CD20+ B cells, and TLS were evaluated on conventional haematoxylin-eosin-stained and immunohistochemical-stained sections of TNBC. We then selected TNBC cases demonstrating the highest and lowest densities of plasma cells, and examined their association with TLS, B cells, as well as immunoglobulin expression using Opal-Vectra multiplex immunofluorescence (IF).

Results: TNBC with high density of plasma cells showed significantly higher numbers of IgG Kappa+ CD38+ cells (p = 0.0089, p < 0.0001), and higher numbers of TLS (p < 0.0001), compared to TNBC with low density of plasma cells. TNBC with high density of plasma cells also showed higher numbers of CD20+ B cells in the tumour core (p < 0.0001), invasive margin (p < 0.0001), as well as stromal (p = 0.015) compartments.

Conclusion: TNBC with high density of plasma cells are associated with higher numbers of IgG Kappa+ CD38+ cells, CD20+ B cells, and TLS. Further studies to characterize the function of plasma cell infiltrates and how they may interact with other tumour-infiltrating lymphocytes and TLS in TNBC may help improve existing immunotherapy strategies.
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http://dx.doi.org/10.1007/s10549-020-05548-yDOI Listing
April 2020

Genomic landscape of lung adenocarcinoma in East Asians.

Nat Genet 2020 02 3;52(2):177-186. Epub 2020 Feb 3.

Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts.
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http://dx.doi.org/10.1038/s41588-019-0569-6DOI Listing
February 2020

The Roles of CD38 and CD157 in the Solid Tumor Microenvironment and Cancer Immunotherapy.

Cells 2019 Dec 20;9(1). Epub 2019 Dec 20.

Institute of Molecular and Cell Biology (IMCB), Agency of Science, Technology and Research (A*STAR), Singapore 138673, Singapore.

The tumor microenvironment (TME) consists of extracellular matrix proteins, immune cells, vascular cells, lymphatics and fibroblasts. Under normal physiological conditions, tissue homeostasis protects against tumor development. However, under pathological conditions, interplay between the tumor and its microenvironment can promote tumor initiation, growth and metastasis. Immune cells within the TME have an important role in the formation, growth and metastasis of tumors, and in the responsiveness of these tumors to immunotherapy. Recent breakthroughs in the field of cancer immunotherapy have further highlighted the potential of targeting TME elements, including these immune cells, to improve the efficacy of cancer prognostics and immunotherapy. CD38 and CD157 are glycoproteins that contribute to the tumorigenic properties of the TME. For example, in the hypoxic TME, the enzymatic functions of CD38 result in an immunosuppressive environment. This leads to increased immune resistance in tumor cells and allows faster growth and proliferation rates. CD157 may also aid the production of an immunosuppressive TME, and confers increased malignancy to tumor cells through the promotion of tumor invasion and metastasis. An improved understanding of CD38 and CD157 in the TME, and how these glycoproteins affect cancer progression, will be useful to develop both cancer prognosis and treatment methods. This review aims to discuss the roles of CD38 and CD157 in the TME and cancer immunotherapy of a range of solid tumor types.
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http://dx.doi.org/10.3390/cells9010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017359PMC
December 2019

Characteristics, behaviour and role of biomarkers in metastatic triple-negative breast cancer.

J Clin Pathol 2020 Mar 28;73(3):147-153. Epub 2019 Sep 28.

Duke-NUS Medical School, Singapore, Singapore

Aims: Characterising the factors responsible for metastatic triple-negative breast cancer (TNBC) is of significant importance, considering its high mortality rate and scant data. In this study, we evaluated the characteristics, clinical behaviour and role of biomarkers (androgen receptor (AR), oestrogen receptor beta (ERβ) and p53) in metastatic TNBC.

Methods: Immunohistochemistry was performed for AR, ERβ and p53 on 125 primary TNBCs with known metastasis and correlated with clinicopathological parameters and outcome. and mRNA profiling was also carried out on 34 tumours from the same series and correlated with outcomes.

Results: In this cohort, grade 3 and pT2 tumours predominated. The most common site for metastasis was the lung and pleura (41, 32.8%), and 15 (12.0%) cases demonstrated metastasis in multiple sites. Among these, 92% of tumours metastasised without preceding local recurrences. Five- and ten-year overall survival (OS) rates were 27% and 7.2%, while 5- and 10- year survival rates after metastasis were 9.6% and 3.2% respectively. AR, ERβ and p53 protein expressions were observed in 16%, 96.8% and 58.1% of tumours, respectively. A combinational phenotype of AR-ERβ+p53+ tumours was associated with poorer OS (HR 1.543, 95%CI 1.030 to 2.310, p=0.035). Higher mRNA levels were significantly associated with favourable OS (p=0.015) and survival after metastasis (p=0.027).

Conclusions: Metastatic TNBC harboured aggressive behaviour and displayed predominantly visceral metastasis with most metastatic events occurring without intervening local recurrences. A combinational phenotype of AR-ERβ+p53+ was significantly associated with poorer OS.
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http://dx.doi.org/10.1136/jclinpath-2019-206078DOI Listing
March 2020

Evaluation of phospho-histone H3 in Asian triple-negative breast cancer using multiplex immunofluorescence.

Breast Cancer Res Treat 2019 Nov 13;178(2):295-305. Epub 2019 Aug 13.

Division of Pathology, Singapore General Hospital, 20 College Road, Singapore, 169856, Singapore.

Purpose: We used multiplex immunofluorescence (mIF) to determine whether mitotic rate represents an independent prognostic marker in triple-negative breast cancer (TNBC). Secondary aims were to confirm the prognostic significance of immune cells in TNBC, and to investigate the relationship between immune cells and proliferating tumour cells.

Methods: A retrospective Asian cohort of 298 patients with TNBC diagnosed from 2003 to 2015 at the Singapore General Hospital was used in the present study. Formalin-fixed, paraffin-embedded breast cancer samples were analysed on tissue microarrays using mIF, which combined phospho-histone H3 (pHH3) expression with cytokeratin (CK) and leukocyte common antigen (CD45) expression to identify tumour and immune cells, respectively.

Results: Multivariate analysis showed that a high pHH3 index was associated with significantly improved overall survival (OS; p = 0.004), but this was not significantly associated with disease-free survival (DFS; p = 0.22). Similarly, multivariate analysis also revealed that a pHH3 positive count of > 1 cell per high-power field in the malignant epithelial compartment was an independent favourable prognostic marker for OS (p = 0.033) but not for DFS (p = 0.250). Furthermore, a high CD45 index was an independent favourable prognostic marker for DFS (p = 0.018), and there was a significant positive correlation between CD45 and pHH3 index (Spearman rank correlation coefficient, 0.250; p < 0.001).

Conclusions: Mitotic rates as determined by pHH3 expression in epithelial cells are significantly associated with improved survival in TNBC. mIF analysis of pHH3 in combination with CK and CD45 could help clinicians in prognosticating patients with TNBC.
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http://dx.doi.org/10.1007/s10549-019-05396-5DOI Listing
November 2019

Pan-cancer analysis connects tumor matrisome to immune response.

NPJ Precis Oncol 2019 22;3:15. Epub 2019 May 22.

1NUS Graduate School for Integrative Sciences & Engineering, National University of Singapore, Singapore, Singapore.

Recent sequencing efforts unveil genomic landscapes of tumor microenvironment. A key compartment in this is the extracellular matrix (ECM) and its related components - matrisome. Yet, little is known about the extent to which matrisome pattern is conserved in progressive tumors across diverse cancer types. Using integrative genomic approaches, we conducted multi-platform assessment of a measure of deregulated matrisome associated with tumor progression, termed as tumor matrisome index (TMI), in over 30,000 patient-derived samples. Combined quantitative analyses of genomics and proteomics reveal that TMI is closely associated with mutational load, tumor pathology, and predicts survival across different malignancies. Interestingly, we observed an enrichment of specific tumor-infiltrating immune cell populations, along with signatures predictive of resistance to immune checkpoint blockade immunotherapy, and clinically targetable immune checkpoints in TMI tumors. B7-H3 emerged as a particularly promising target for anti-tumor immunity in these tumors. Here, we show that matrisomal abnormalities could represent a potential clinically useful biomarker for prognostication and prediction of immunotherapy response.
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http://dx.doi.org/10.1038/s41698-019-0087-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531473PMC
May 2019

Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates.

Nature 2018 05 16;557(7706):575-579. Epub 2018 May 16.

Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), Singapore, Singapore.

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8 TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein-Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8 TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8 TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39 CD8 TILs. Furthermore, frequencies of CD39 expression among CD8 TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.
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http://dx.doi.org/10.1038/s41586-018-0130-2DOI Listing
May 2018

Using computer assisted image analysis to determine the optimal Ki67 threshold for predicting outcome of invasive breast cancer.

Oncotarget 2018 Feb 5;9(14):11619-11630. Epub 2018 Feb 5.

Division of Pathology, Singapore General Hospital, Singapore.

Background: Ki67 positivity in invasive breast cancers has an inverse correlation with survival outcomes and serves as an immunohistochemical surrogate for molecular subtyping of breast cancer, particularly ER positive breast cancer. The optimal threshold of Ki67 in both settings, however, remains elusive. We use computer assisted image analysis (CAIA) to determine the optimal threshold for Ki67 in predicting survival outcomes and differentiating luminal B from luminal A breast cancers.

Methods: Quantitative scoring of Ki67 on tissue microarray (TMA) sections of 440 invasive breast cancers was performed using Aperio ePathology ImmunoHistochemistry Nuclear Image Analysis algorithm, with TMA slides digitally scanned via Aperio ScanScope XT System.

Results: On multivariate analysis, tumours with Ki67 ≥14% had an increased likelihood of recurrence (HR 1.941, p=0.021) and shorter overall survival (HR 2.201, p=0.016). Similar findings were observed in the subset of 343 ER positive breast cancers (HR 2.409, p=0.012 and HR 2.787, p=0.012 respectively). The value of Ki67 associated with ER+HER2-PR<20% tumours (Luminal B subtype) was found to be <17%.

Conclusion: Using CAIA, we found optimal thresholds for Ki67 that predict a poorer prognosis and an association with the Luminal B subtype of breast cancer. Further investigation and validation of these thresholds are recommended.
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http://dx.doi.org/10.18632/oncotarget.24398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837769PMC
February 2018

An automated staining protocol for seven-colour immunofluorescence of human tissue sections for diagnostic and prognostic use.

Pathology 2018 Apr 9;50(3):333-341. Epub 2018 Feb 9.

Department of Anatomical Pathology, Singapore General Hospital, Singapore; Duke-NUS Medical School, Singapore. Electronic address:

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection and quantification of the expression of up to six markers, plus a nuclear counterstain, on a single tissue section. Recent studies have shown the potential for mIF to advance our understanding of complex disease processes, including cancer. It is important that the technique be standardised and validated to facilitate its transition into clinical use. Traditional approaches to mIF rely on manual processing of sections, which is time-consuming and a source of significant variation between samples/individuals. Here we determined if an automated diagnostic tissue stainer could be used for mIF incorporating tyramide signal amplification (TSA), and how the final image quality compared with sections stained semi-automatically or manually. Using tissue microarrays of fixed human breast tumour sections, we observed comparable antibody labelling between the diagnostic autostainer and manual technique. The diagnostic autostainer produced higher signal intensity with similar spectral unmixing efficiency. We also found that microwave treatment for antibody stripping during TSA labelling could be replaced by the heating option incorporated within the diagnostic-use autostainer. These data show that diagnostic autostainers used for traditional immunohistochemistry protocols can be readily adapted to achieve rapid preparation of high-quality sections using a TSA method for clinical mIF.
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http://dx.doi.org/10.1016/j.pathol.2017.11.087DOI Listing
April 2018

PD-1 expression on dendritic cells suppresses CD8 T cell function and antitumor immunity.

Oncoimmunology 2016 Mar 31;5(3):e1085146. Epub 2015 Aug 31.

Genomics, Genetics and Biology Innovation Pole (PoloGGB), Piazza Gambuli, Edificio D, 3° Piano, Perugia, Italy; A. Menarini Biomarkers Singapore Pte Ltd, 30 Pasir Panjang Road, #08-32, Mapletree Business City, Singapore.

Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8 T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8 T cell proliferation . In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8 effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1.
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http://dx.doi.org/10.1080/2162402X.2015.1085146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839350PMC
March 2016

Activation and regulation of interferon-β in immune responses.

Immunol Res 2012 Sep;53(1-3):25-40

Laboratory of Gene Regulation and Inflammation, Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #04 Immunos, Biopolis, Singapore.

Interferons (IFNs) were discovered more than half a century ago, and extensive research has since identified multifarious roles for type I IFN in human immune responses. Here, we review the functions of IFN-β in innate and adaptive immunity. We also discuss the activation and influence of IFN-β on myeloid cell types, including monocytes and dendritic cells, as well as address the effects of IFN-β on T cells and B cells. Findings from our own laboratory, which explores the molecular mechanisms of IFN-β activation by LPS and viruses, as well as from other groups investigating the regulation of IFN-β by viral proteins and endogenous factors are described. The effects of post-translational modifications of the interferon regulatory factor (IRF)-3 on IFN-β induction are also highlighted. Many unanswered questions remain concerning the regulation of the type I IFN response in inflammation, especially the role of transcription factors in the modulation of inflammatory gene expression, and these questions will form the basis for exciting avenues of future research.
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http://dx.doi.org/10.1007/s12026-012-8293-7DOI Listing
September 2012