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

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.

Nosocomial infections among COVID-19 patients: an analysis of intensive care unit surveillance data.

Surveillance of nosocomial infections, like catheter-associated urinary tract infection (CAUTI), central line-associated bloodstream infection, possible ventilator-associated pneumonia and secondary bloodstream infections were observed to study the impact of COVID-19 outbreak in ICUs from Tan Tock Seng Hospital and National Centre for Infectious Diseases, Singapore between February and June 2020. Higher nosocomial infection rates were observed in COVID-19 patients, although it was not statistically significant. Moreover, COVID-19 patients seem to be more predisposed to CAUTI despite a higher proportion of non-COVID-19 patients having urinary catheters. Thus, continued vigilance to ensure adherence to IPC measures is needed.

SRSF1 mediates cytokine-induced impaired imatinib sensitivity in chronic myeloid leukemia.

Patients with chronic myeloid leukemia (CML) who are treated with tyrosine kinase inhibitors (TKIs) experience significant heterogeneity regarding depth and speed of responses. Factors intrinsic and extrinsic to CML cells contribute to response heterogeneity and TKI resistance. Among extrinsic factors, cytokine-mediated TKI resistance has been demonstrated in CML progenitors, but the underlying mechanisms remain obscure. Using RNA-sequencing, we identified differentially expressed splicing factors in primary CD34+ chronic phase (CP) CML progenitors and controls. We found SRSF1 expression to be increased as a result of both BCR-ABL1- and cytokine-mediated signaling. SRSF1 overexpression conferred cytokine independence to untransformed hematopoietic cells and impaired imatinib sensitivity in CML cells, while SRSF1 depletion in CD34+ CP CML cells prevented the ability of extrinsic cytokines to decrease imatinib sensitivity. Mechanistically, PRKCH and PLCH1 were upregulated by elevated SRSF1 levels, and contributed to impaired imatinib sensitivity. Importantly, very high SRSF1 levels in the bone marrow of CML patients at presentation correlated with poorer clinical TKI responses. In summary, we find SRSF1 levels to be maintained in CD34+ CP CML progenitors by cytokines despite effective BCR-ABL1 inhibition, and that elevated levels promote impaired imatinib responses. Together, our data support an SRSF1/PRKCH/PLCH1 axis in contributing to cytokine-induced impaired imatinib sensitivity in CML.

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

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 (ERß) and p53) in metastatic TNBC. METHODS: Immunohistochemistry was performed for AR, ERß and p53 on 125 primary TNBCs with known metastasis and correlated with clinicopathological parameters and outcome. AR and p53 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, ERß and p53 protein expressions were observed in 16%, 96.8% and 58.1% of tumours, respectively. A combinational phenotype of AR-ERß+p53+ tumours was associated with poorer OS (HR 1.543, 95%CI 1.030 to 2.310, p=0.035). Higher AR 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-ERß+p53+ was significantly associated with poorer OS.

Multiplex immunohistochemistry/immunofluorescence (mIHC/IF) for PD-L1 testing in triple-negative breast cancer: a translational assay compared with conventional IHC.

BACKGROUND: Programmed death-ligand 1 (PD-L1) monoclonal antibody therapy has recently gained approval for treating metastatic triple-negative breast cancer (TNBC) -, in particular in the PD-L1+ patient subgroup of the recent IMpassion130 trial. The SP142 PD-L1 antibody clone was used as a predictive assay in this trial, but this clone was found to be an outlier in previous harmonisation studies in lung cancer. AIMS: To address the comparability of PD-L1 clones in TNBC, we evaluated the concordance between conventional immunohistochemistry (IHC) and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) that allowed simultaneous quantification of three different PD-L1 antibodies (22C3, SP142 and SP263). METHODS: Our cohort comprised 25 TNBC cases, 12 non-small-cell lung carcinomas and 8 other cancers. EpCAM labelling was used to distinguish tumour cells from immune cells. RESULTS: Moderate-to-strong correlations in PD-L1 positivity were found between results obtained through mIHC/IF and IHC. Individual concordance rates in the study ranged from 67% to 100%, with Spearman's rank correlation coefficient values up to 0.88. CONCLUSIONS: mIHC/IF represents a promising tool in the era of cancer immunotherapy, as it can simultaneously detect and quantify PD-L1 labelling with multiple antibody clones, and allow accurate evaluation of tumour and immune cells. Clinicians and pathologists require this information to predict patient response to anti-PD-1/PD-L1 therapy. The adoption of this assay may represent a significant advance in the management of therapeutically challenging cancers. Further analysis and assay harmonisation are essential for translation to a routine diagnostic setting.

Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer.

The role of programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) in triple negative breast cancer (TNBC) remains to be fully understood. In this study, we investigated the role of PD-1 as a prognostic marker for TNBC in an Asian cohort (n = 269). Samples from patients with TNBC were labeled with antibodies against PD-L1 and PD-1, and subjected to NanoString assays to measure the expression of immune-related genes. Associations between disease-free survival (DFS), overall survival (OS) and biomarker expression were investigated. Multivariate analysis showed that tumors with high PD-1+ immune infiltrates harbored significantly increased DFS, and this increase was significant even after controlling for clinicopathological parameters (HR 0.95; P = 0.030). In addition, the density of cells expressing both CD8 and PD-1, but not the density of CD8-PD-1+ immune infiltrates, was associated with improved DFS. Notably, this prognostic significance was independent of clinicopathological parameters and the densities of total CD8+ cell (HR 0.43, P = 0.011). At the transcriptional level, high expression of PDCD1 within the tumor was significantly associated with improved DFS (HR 0.38; P = 0.027). In line with these findings, high expression of IFNG (HR 0.38; P = 0.001) and IFN signaling genes (HR 0.46; p = 0.027) was also associated with favorable DFS. Inclusion of PD-1 immune infiltrates and PDCD1 gene expression added significant prognostic value for DFS (ΔLRχ2 = 6.35; P = 0.041) and OS (ΔLRχ2 = 9.53; P = 0.008), beyond that provided by classical clinicopathological variables. Thus, PD-1 mRNA and protein expression status represent a promising, independent indicator of prognosis in TNBC.

Expression of CD38 on Macrophages Predicts Improved Prognosis in Hepatocellular Carcinoma.

Background: CD38 is involved in the adenosine pathway, which represents one of the immunosuppressive mechanisms in cancer. CD38 is broadly expressed across immune cell subsets, including human macrophages differentiated in vitro from monocytes, but expression by tissue-resident macrophages remains to be demonstrated. Methods: Tissue samples were obtained from 66 patients with hepatocellular carcinoma (HCC) from Singapore and analyzed using immunohistochemistry. Tumor-infiltrating leukocytes (TILs) were further examined using DEPArray™, and the phenotype of freshly isolated TILs was determined using flow cytometry. Results: CD38 was frequently co-expressed with the macrophage-specific marker CD68. CD38+CD68+ macrophage density was associated with improved prognosis after surgery, while total CD68+ macrophage density was associated with poor prognosis. DEPArray™ analysis revealed the presence of large (>10 µm), irregularly shaped CD45+CD14+ cells that resembled macrophages, with concurrent CD38+ expression. Flow cytometry also revealed that majority of CD14+HLA-DR+ cells expressed CD38. Conclusion: CD38 expression was clearly demonstrated on human macrophages in an in vivo setting. The positive association identified between CD38+ macrophage density and prognosis may have implications for routine diagnostic work.

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

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.

High Densities of Tumor-Associated Plasma Cells Predict Improved Prognosis in Triple Negative Breast Cancer.

Breast cancer is the most common malignancy affecting women, but the heterogeneity of the condition is a significant obstacle to effective treatment. Triple negative breast cancers (TNBCs) do not express HER2 or the receptors for estrogen or progesterone, and so often have a poor prognosis. Tumor-infiltrating T cells have been well-characterized in TNBC, and increased numbers are associated with better outcomes; however, the potential roles of B cells and plasma cells have been large. Here, we conducted a retrospective correlative study on the expression of B cell/plasma cell-related genes, and the abundance and localization of B cells and plasma cells within TNBCs, and clinical outcome. We analyzed 269 TNBC samples and used immunohistochemistry to quantify tumor-infiltrating B cells and plasma cells, coupled with NanoString measurement of expression of immunoglobulin metagenes. Multivariate analysis revealed that patients bearing TNBCs with above-median densities of CD38+ plasma cells had significantly better disease-free survival (DFS) (HR = 0.44; 95% CI 0.26-0.77; p = 0.004) but not overall survival (OS), after adjusting for the effects of known prognostic factors. In contrast, TNBCs with higher immunoglobulin gene expression exhibited improved prognosis (OS p = 0.029 and DFS p = 0.005). The presence of B cells and plasma cells was positively correlated (p < 0.0001, R = 0.558), while immunoglobulin gene IGKC, IGHM, and IGHG1 mRNA expression correlated specifically with the density of CD38+ plasma cells (IGKC p < 0.0001, R = 0.647; IGHM p < 0.0001, R = 0.580; IGHG1 p < 0.0001, R = 0.655). Interestingly, after adjusting the multivariate analysis for the effect of intratumoral CD38+ plasma cell density, the expression levels of all three genes lost significant prognostic value, suggesting a biologically important role of plasma cells. Last but not least, the addition of intratumoral CD38+ plasma cell density to clinicopathological features significantly increased the prognostic value for both DFS (ΔLRχ2 = 17.28, p = 1.71E-08) and OS (ΔLRχ2 = 10.03, p = 6.32E-08), compared to clinicopathological features alone. The best combination was achieved by integrating intratumoral CD38+ plasma cell density and IGHG1 which conferred the best added prognostic value for DFS (ΔLRχ2 = 27.38, p = 5.22E-10) and OS (ΔLRχ2 = 21.29, p = 1.03E-08). Our results demonstrate that the role of plasma cells in TNBC warrants further study to elucidate the relationship between their infiltration of tumors and disease recurrence.