PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer

BackgroundThe prognostic value of tumor‐associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c⁺ DC co–expressing the immunoinhibitory molecule PD‐L1 and their spatial relationship with CD8⁺ T‐cells in patients treated for stage III colon cancer.MethodsTissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD‐L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan‐Meier estimates and Cox regression were used to assess survival.ResultsIntratumoral CD8⁺ cell density (HR = .52, 95% confidence interval [CI] .33‐.83, P = .007), stromal CD11c⁺ cell density (HR = .52, 95% CI .33‐.83, P = .006), intratumoral CD11c⁺PD‐L1⁺ cell density (HR = .57, 95% CI .35‐.92, P = .021), and stromal CD11c⁺PD‐L1⁺ cell density (HR = .48, 95% CI .30‐.77, P = .003) on leading‐edge cores were all significantly associated with good survival. CD8⁺ cell density was positively correlated with both CD11c⁺ cell density and CD11c⁺PD‐L1⁺ cell density in tumor epithelium and stromal compartments.ConclusionHere we showed that PD‐L1‐expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically “hot” tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor‐associated DC may help to further elucidate their prognostic value.     

De novo diffuse large B‐cell lymphoma with a CD20 immunohistochemistry‐positive and flow cytometry‐negative phenotype: Molecular mechanisms and correlation with rituximab sensitivity

CD20 is expressed in most B-cell lymphomas and is a critical molecular target of rituximab. Some B-cell lymphomas show aberrant CD20 expression, and rituximab use in these patients is controversial. Here we show both the molecular mechanisms and the clinical significance of de novo diffuse large B-cell lymphomas (DLBCL) that show a CD20 immunohistochemistry (IHC)-positive and flow cytometry (FCM)- negative (IHC[+]/FCM[−]) phenotype. Both IHC and FCM using anti-CD20 antibodies L26 and B1, respectively, were analyzed in 37 of the 106 cases of de novo DLBCL; 8 (22%) of these cases were CD79a(+)/CD20(+) with IHC and CD19(+)/CD20(−) with FCM. CD20 (MS4A1) mRNA expression was significantly lower in IHC(+)/FCM(−) cells than in IHC(+)/FCM(+) cells (P = 0.0005). No genetic mutations were detected in MS4A1 promoter and coding regions. Rituximab-mediated cytotoxicity in the CDC assay using IHC(+)/FCM(−) primary cells was significantly lower than in IHC(+)/FCM(+) cells (P < 0.05); however, partial effectiveness was confirmed. FCM using rituximab detected CD20 more efficiently than B1. No significant difference was observed between IHC(+)/FCM(−) and IHC(+)/FCM(+) patients in overall survival (P = 0.664). Thus, lower expression of CD20 mRNA is critical for the CD20 IHC(+)/FCM(−) phenotype. Lower CD20 expression with FCM does not rule out rituximab use in these patients if expression is confirmed with IHC. FCM using rituximab may be more informative than B1 for predicting rituximab effectiveness in IHC(+)/FCM(−) cases.     

Clinical relevance of PD-1 positive CD8 T-cells in gastric cancer

Background

We evaluated the relevance of PD-1⁺CD8⁺ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME).

Methods

Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analyzed bulk RNAseq GC datasets from TCGA, the “3G” chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analyzed single-cell RNAseq performed on GCs.

Results

In the discovery cohort of 350 GCs, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.822, p = 0.042). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B⁺] (r = 0.714, p < 0.001) and proliferative [Ki-67⁺] (r = 0.798, p < 0.001) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8A expression levels had improved OS when treated with immunotherapy (HR 0.117, p = 0.036) and chemotherapy (HR 0.475, p = 0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p < 0.0001), while macrophage proportions were lower (7% vs 11%, p < 0.0001) in CD8PD-1_high compared to CD8PD-1_low tumors.

Conclusion

This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1⁺CD8⁺ T-cells being associated with improved OS. CD8PD-1_high tumors have distinct features of an immunologically active, T-cell inflamed TME.

     

Circulating and tumor-infiltrating Tim-3 in patients with colorectal cancer

T-cell exhaustion represents a progressive loss of T-cell function. The inhibitory receptor PD-1 is known to negatively regulate CD8⁺ T cell responses directed against tumor antigen, but the blockades of PD-1 pathway didn''t show the objective responses in patients with colorectal cancer (CRC). Thus, further exploring the molecular mechanism responsible for inducing T-cell dysfunction in CRC patients may reveal effective strategies for immune therapy. This study aims to characterize co-inhibitory receptors on T cells in CRC patients to identify novel targets for immunotherapy. In this study, peripheral blood samples from 20 healthy controls and 54 consented CRC patients, and tumor and matched paraneoplastic tissues from 7 patients with advanced CRC, subjected to multicolor flow cytometric analysis of the expression of PD-1 and Tim-3 receptors on CD8⁺ T cells. It was found that CRC patients presented with significantly higher levels of circulating Tim-3⁺PD-1⁺CD8⁺ T cells compared to the healthy controls (medians of 3.12% and 1.99%, respectively, p = 0.0403). A similar increase of Tim-3⁺PD-1⁺CD8⁺ T cells was also observed in the tumor tissues compared to paraneoplastic tussues. Tim-3⁺PD-1⁺CD8⁺ T cells in tumor tissues produced even less cytokine than that in paraneoplastic tissues. Functional ex vivo experiments showed that Tim-3⁺PD-1⁺CD8⁺ T cells produced significantly less IFN-γ than Tim-3⁻PD-1⁻CD8⁺ T cells, followed by Tim-3⁺PD-1⁻CD8⁺ T cells, and Tim-3⁻PD-1⁺CD8⁺ T cells, indicating a stronger inhibition of IFN-γ production of Tim-3⁺CD8⁺ T cells. It is also found in this study that Tim-3⁺PD-1⁺CD8⁺ T cell increase in circulation was correlated with clinical cancer stage but not histologic grade and serum concentrations of cancer biomarker CEA. Our results indicate that upregulation of the inhibitory receptor Tim-3 may restrict T cell responses in CRC patients, and therefore blockage of Tim-3 and thus restoring T cell responses may be a potential therapeutic approach for CRC patients.     

Concurrent High PD-L1 Expression and CD8+ Immune Cell Infiltration Predict PD-1 Blockade Efficacy in Advanced EGFR-Mutant NSCLC Patients

ObjectivesThe effectiveness of PD-1 blockade therapy in advanced epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) is limited. We investigated whether patient characteristics, PD-L1 expression, and immune cell (IC) status in the tumor microenvironment (TME) were associated with PD-1 blockade therapy outcomes.Materials and methodsWe retrospectively reviewed patients with advanced EGFR-mutant NSCLC treated with PD-1 blockade (nivolumab or pembrolizumab) between January 2016 and March 2018. The PD-L1 expression tumor proportion score (TPS) and types and distribution of ICs (CD8, PD-1, CD204, tumoral, and stromal) in the TME were analyzed.ResultsAmong 57 EGFR-mutant NSCLC patients treated with PD-1 blockade, 39 patients had sufficient tissues for analyzing the TME. The overall response rate (ORR) of PD-1 blockade was 12.3%. Only tumoral CD8 positive (CD8⁺) IC status was significantly associated with the response (median tumoral CD8⁺ICs: 299/mm² vs. 115/mm², P < .01). Among the 6 patients with concurrent high PD-L1 expression (TPS: ≥ 50%)/high tumoral CD8⁺ ICs (≥ 205/mm²), 5 (83.3%) showed a response and a significantly longer progression-free survival (PFS) (PFS: 9.4M vs. 1.8M, P < .01). In contrast, none of the 7 patients with high PD-L1 expression/low tumoral CD8⁺ICs (<205/mm²) showed a response.ConclusionConcurrent high PD-L1 expression and high tumoral CD8⁺ ICs could predict the response and longer PFS of PD-1 blockade therapy in EGFR-mutant patients.     

CD4+ and CD8+ T cells in sentinel nodes exhibit distinct pattern of PD-1, CD69, and HLA-DR expression compared to tumor tissue in oral squamous cell carcinoma

Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti-programmed cell death protein 1 (PD-1) treatment is predominantly studied in tumor-infiltrating lymphocytes (TILs). Its impact on PD-1 expressing cells in tumor-draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD-1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD-1, CD69, and HLA-DR on CD4⁺ and CD8⁺ T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD-1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD-1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD-1 negative TILs expressed high levels of human leukocyte antigen – DR isotype and CD69. In contrast, PD-1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3⁺ PD-1⁺ cells in tumor-draining lymph nodes had significantly lower disease-free and overall survival rates (log-rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD-1 levels in TDLNs correspond with survival and potentially with response to anti-PD-1 therapy. Such knowledge may ultimately help guide anti-PD-1 treatment.     

Programmed death-1 ligand 1 and 2 are highly expressed in pleomorphic carcinomas of the lung: Comparison of sarcomatous and carcinomatous areas

Pleomorphic carcinoma (PC) of the lung is a rare type of poorly differentiated non-small cell lung carcinoma (NSCLC) that belongs to sarcomatoid carcinoma (SC). It exhibits aggressive behaviour and resistance to chemotherapy and radiotherapy. Recently, immunotherapy targeting the programmed death-1 (PD-1)/PD ligand 1 (PD-L1) pathway has demonstrated favourable clinical outcomes in NSCLC. However, the expression patterns of PD-1-related molecules in pulmonary PC remain elusive.PD-L1 and PD-L2 expression was estimated in 41 cases of PC using immunohistochemistry. CD8⁺ and PD-1⁺ tumour-infiltrating lymphocytes (TILs) were also evaluated.PD-L1 and PD-L2 were highly expressed in pulmonary PCs (90.2% [37/41)]; 87.8% [36/41]). The amount of CD8⁺ or PD-1⁺ TILs and the ratio of PD-1⁺/CD8⁺ TILs in PC were higher in males, smokers and older patients. PD-L1-positive PCs were infiltrated by higher numbers of CD8⁺ TILs compared to PD-L1-negative cases (P = 0.006). Of note, PD-L1 expression in pulmonary PCs was significantly higher in sarcomatous areas than in the carcinomatous portion (P = 0.006). PC patients with a high ratio of PD-1⁺/CD8⁺ TILs showed a shorter progression-free survival (P = 0.036), whereas PD-L1 and PD-L2 expression had no prognostic implications.Our study demonstrates that pulmonary PCs very frequently express PD-L1 and PD-L2. Moreover, their expression is higher in sarcomatous cells than in carcinomatous areas. Thus, targeting the PD-1/PD-L1 pathway may represent a potential therapeutic candidate for this aggressive tumour.     

Increased expression of immunosuppressive molecules on intratumoral and circulating regulatory T cells in non-small-cell lung cancer patients

The expression patterns of immunosuppressive molecules on regulatory T (Treg) cells have not been elucidated in non-small-cell lung cancer (NSCLC) patients. In this study, a total of 88 patients including 53 patients with NSCLC, 17 patients with lung non-malignant diseases, and 18 healthy volunteers were enrolled. Increased number of total CD4⁺CD25⁺FoxP3⁺ Treg cells and elevated expressions on the surface of several inhibitory molecules including CTLA-4, LAG-3 and PD-1 have been observed in the peripheral blood of NSCLC patients. We found that intratumoral Treg cells from NSCLC patients express the highest levels of co-inhibitory molecules compared to Treg cells isolated from tumor adjacent tissues or from peripheral blood of cancer patients, which is in consistent with the enhanced immunosuppressive function of these co-inhibitory molecules. Moreover, the number of Treg cells and their functional surface molecules increased during the progression of lung cancer. Elevated plasma levels of TGF-β and IL-10 in NSCLC patients were also observed in NSCLC patients compared to that in healthy volunteers. Our findings further support the role of Treg cells in the tumor microenvironments in NSCLC patients.     

Mobilization of peripheral blood stem cells using regimen combining docetaxel with granulocyte colony-stimulating factor in breast cancer patients

Objective:To evaluate the effectiveness and safety of the mobilization of peripheral blood hematopoietic stem cells by combining docetaxel with granulocyte colony-stimulating factor(G-CSF) in breast cancer patients.Methods:A total of 57 breast cancer patients were treated with docetaxel 120 mg/m2.When the white blood cell(WBC) count decreased to 1.0×109/L,patients were given G-CSF 5-g/kg daily by subcutaneous injection until the end of apheresis.Peripheral blood mononuclear cells(MNC) were isolated by Cobe Spectra Apheresis System.The percentage of CD34+ cell was assayed by flow cytometry.Results:At a median 6 of days(range 3-8) after the administration of docetaxel,the median WBC count decreased to 1.08×109/L(range 0.20-2.31).The median duration of G-CSF mobilization was 3 days(range 2-7).The MNC collection was conducted 8-12 days(median 10 days) after docetaxel treatment.The median MNC was 5.35×108/kg(range 0.59-14.07),the median CD34+ cell count was 2.43×106/kg(range 0.16-16.69).The CD34+ cell count was higher than 1.00×106/kg in 47 of 57 cases(82.46%) and higher than 2.00×106/kg in 36 cases(63.16%).The CD34+ cell count was higher than 2.00×106/kg in 27 collections(23.68%).The MNC count and the CD34+ cell count were correlated with the bottom of WBC after docetaxel chemotherapy(r=0.364,0.502,P=0.005,0.000).The CD34+ cell count was correlated with the MNC count(r=0.597,P=0.000).The mobilization and apheresis were well tolerated in all patients.Mild perioral numbness and numbness of hand or feet were observed in 3 cases.No serious adverse events were reported.Conclusion:Mobilization of peripheral blood hematopoietic stem cell by combining docetaxel with G-CSF was effective and safety in breast cancer patients.     

Immune Biomarkers in Blood from Sarcoma Patients: A Pilot Study

The main role of the host immune system is to identify and eliminate cancer cells, which is a complex process, but it is not a fail-safe mechanism. Many sarcoma patients succumb to this disease despite treatments rendered. The aim of this pilot study was to compare the levels of CD4⁺ T-cells, T-regulatory (Treg) cells, and cytokines such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-17A (IL-17A), and transforming growth factor-beta-1 (TGF-β1) in peripheral blood leukocytes of sarcoma patients and healthy controls. For gene expression studies, total ribonucleic acid (RNA) was extracted from peripheral blood leukocytes and genes that were differentially regulated in peripheral blood leukocytes of sarcoma patients compared with healthy controls were determined using a commercial T-helper cell differentiation quantitative polymerase chain reaction (qPCR) array. Flow cytometer analysis was performed on blood samples from 26 sarcoma patients and 10 healthy controls to identify the levels of CD4⁺ T-cells and T-reg cells. The level of cytokines in plasma and culture supernatant were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. A marked reduction in the percentage of CD4⁺ T-cells (p = 0.037) and levels of TNF-α (p = 0.004) and IFN-γ (0.010) was observed in sarcoma patients. Gene expression analysis showed five genes (homeobox A10 (HOXA10), GATA binding protein 3 (GATA3), prostaglandin D2 receptor 2 (PTGDR2), thymocyte selection associated high mobility group box (TOX), and C-C motif chemokine receptor 3 (CCR3)) were dysregulated (p < 0.05) in sarcoma patients. This study suggests that T-helper-1 immune responses are reduced in sarcoma patients.     

Immune landscape of vulvar cancer patients treated with surgery and adjuvant radiotherapy revealed restricted T cell functionality and increased IL-17 expression associated with cancer relapse

For vulvar cancers, radiotherapy is targeting cancer cells, but also affects the host immune system. As this may affect treatment outcome, in this prospective study, we characterized the individual T cell immune milieu induced by surgery and adjuvant radio +/− chemotherapy (aRT) systemically in the blood of vulvar cancer patients and found increased frequencies of Interleukin (IL)-17-producing CD4⁺ and CD8⁺ T cells after aRT while frequencies of Th1 and perforin-producing CD8⁺ killer cells were strongly diminished. Phenotypic characterization revealed enhanced expression of the ectonucleotidase CD39 on Th17 and Tc17 cells as well as CD8⁺ perforin⁺ cells after aRT. Furthermore, the aRT cohort exhibited increased proportions of Programmed Cell Death Protein (PD-1) expressing cells among Th1 and CD8⁺ perforin⁺ cells, but not among Th17 and Tc17 cells. High post-therapeutic levels of Th17 and Tc17 cells and low proportions of Th1 and CD8⁺ perforin⁺ cells expressing PD-1 was associated with reduced recurrence free survival on follow-up. In conclusion, our study defines individual therapy-induced changes in the cellular immune milieu of patients and their association with cancer relapse. Our results may help to explain differences in the individual courses of disease of vulvar cancer patients and suggest PD-1 and IL-17 as targets for immunotherapy in vulvar cancer.     

Immunotherapies targeting neoantigens are effective in PD-1 blockade-resistant tumors

Only a small fraction of tumor-infiltrating lymphocytes can specifically recognize and attack cancer cells in PD-1/PD-L1 blockade therapy. Here, we investigate approaches to expand the neoantigen-specific CD8⁺ T cells to overcome the difficulties in treating PD-1/PD-L1 blockade-resistant tumors. Mutation-associated neoepitopes of murine nonsmall cell lung cancer ASB-XIV were estimated by whole-exome and RNA sequencing and predicted by MHC-I binding affinity (FPKM >1) in silico. Using ASB-XIV-specific CD8⁺ T cells, we screened a panel of 257 neoepitope peptides derived from ASB-XIV missense and indel mutations. Mutated Phf3 peptide (mPhf3) was successfully identified as an immunogenic neoepitope. Prophylactic mPhf3-DC vaccination inhibited ASB-XIV tumor growth through CD8⁺ T cell-mediated antitumor immunity. Combining the mPhf3-DC vaccine and anti-PD-1 treatment elicited robust antitumor activity through the induction of mPhf3-specific CD8⁺ T cells in the tumor microenvironment. Furthermore, the adoptive transfer of mPhf3-specific CD8⁺ T cells eradicated ASB-XIV tumors. Likewise, the combination of mutated Cdt1 peptide (mCdt1)-DC vaccine and anti-PD-1 treatment or adoptive transfer of mCdt1-specific CD8⁺ T cells also led to significant regression of PD-1 blockade-resistant murine gastric YTN16 tumors. In conclusion, a novel immunogenic neoepitope of ASB-XIV was identified for immunotherapy targeting neoantigens. Identification of immunogenic neoantigens can extend the therapeutic strategies by increasing the frequency of neoantigen-specific T cells, even for PD-1/PD-L1 blockade-resistant tumors.     

18F‐fluorodeoxyglucose uptake in PET is associated with the tumor microenvironment in metastatic lymph nodes and prognosis in N2 lung adenocarcinoma

Positron emission tomography is a useful technique for diagnosing lymph node (LN) metastasis. This study aimed to elucidate the association between fluorodeoxyglucose accumulation and the microenvironment in metastatic LNs in lung adenocarcinoma. We retrospectively analyzed 62 patients with surgically resected pathological N2 lung adenocarcinoma who underwent preoperative PET. The maximum standardized uptake value (SUV_max) in the metastatic LNs was measured. Lymph node specimens were immunohistochemically analyzed for CD8⁺, FoxP3⁺, and CD79a⁺ lymphocytes, CD204⁺ tumor‐associated macrophages (TAMs), and alpha‐smooth muscle actin‐positive cancer‐associated fibroblasts (αSMA⁺ CAFs). We compared the clinicopathologic and immunohistochemical characteristics between two groups with high and low LN SUV_max. Using novel 3D hybrid spheroid models, we investigated the change in invasiveness of cancer cells in the presence of CAFs. In the multivariate analyses, LN SUV_max was an independent prognostic factor. The overall survival in the LN SUV_max high group was significantly worse than in the low group (P = .034). In the LN SUV_max high group, metastatic cancer cell invasion of extranodal tissue was more frequent (P = .005) and the number of CD204⁺ TAMs and αSMA⁺ CAFs in metastatic LNs was significantly higher than in the low group (P < .001 and P = .002, respectively). Hybrid spheroid models revealed that cancer cells coexisting with CAFs were more invasive than those without CAFs. Our results indicated a strong association between LN SUV_max and poor prognosis in patients with N2 lung adenocarcinoma. Moreover, LN SUV_max was suggested to be associated with the presence of tumor‐promoting stromal cells in metastatic LNs.     

Flow cytometric immunophenotypic characteristics of 36 cases of plasma cell leukemia

Prospective flow cytometric analysis of antigens expression on bone marrow and peripheral blood plasma cells of 36 plasma cell leukemia (PCL) patients enabled to establish the following immunophenotype of leukemic plasma cell: CD38⁺⁺, CD138⁺, CD54⁺, CD49d⁺, CD29⁺, CD44⁺, CD126⁺, CD19⁻, CD45⁻. In one-third of patients PCL cells express CD56, CD71 and CD117. Expression of CD54 on plasma cells was higher as compared to expression of adhesion molecules CD11a, CD18 and CD11b (p < 0.01). Expression of CD18, CD11a, CD11b was lower on bone marrow and higher on peripheral blood cells. In conclusion, impaired expression of adhesion molecules such as CD11a/CD18 or CD56 may explain hematogenic dissemination characterizing PCL.     

Up‐regulation of proliferative and migratory genes in regulatory T cells from patients with metastatic castration‐resistant prostate cancer

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor‐induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome‐wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration‐resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin‐2 (IL‐2) and transforming growth factor‐β (TGF‐β) pathways. Studies revealed that the levels of expression of genes responsible for T‐cell proliferation (C‐FOS, C‐JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4⁺CD25^highCD127^– Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.     

Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice

Ex vivo expansion of CD8⁺ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8⁺ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8⁺ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8⁺ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2×10⁶ CD8⁺ T-cells and achieved the same level of tumor growth inhibition as that of 5×10⁶ CD8⁺ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-β, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8⁺ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8⁺ T-cells which also stay longer.     

Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer

BackgroundImmune checkpoint blockade with Programmed cell death 1 (PD-1)/PD-L1 inhibitors has been effective in various malignancies and is considered as a standard treatment modality for patients with non-small-cell lung cancer (NSCLC). However, emerging evidence show that PD-1/PD-L1 blockade can lead to hyperprogressive disease (HPD), a flair-up of tumor growth linked to dismal prognosis. This study aimed to evaluate the incidence of HPD and identify the determinants associated with HPD in patients with NSCLC treated with PD-1/PD-L1 blockade.Patients and methodsWe enrolled patients with recurrent and/or metastatic NSCLC treated with PD-1/PD-L1 inhibitors between April 2014 and November 2018. Clinicopathologic variables, dynamics of tumor growth, and treatment outcomes were analyzed in patients with NSCLC who received PD-1/PD-L1 blockade. HPD was defined according to tumor growth kinetics (TGK), tumor growth rate (TGR), and time to treatment failure (TTF). Immunophenotyping of peripheral blood CD8⁺ T lymphocytes was conducted to explore the potential predictive biomarkers of HPD.ResultsA total of 263 patients were analyzed. HPD was observed in 55 (20.9%), 54 (20.5%), and 98 (37.3%) patients according to the TGK, TGR, and TTF. HPD meeting both TGK and TGR criteria was associated with worse progression-free survival [hazard ratio (HR) 4.619; 95% confidence interval (CI) 2.868–7.440] and overall survival (HR, 5.079; 95% CI, 3.136–8.226) than progressive disease without HPD. There were no clinicopathologic variables specific for HPD. In the exploratory biomarker analysis with peripheral blood CD8⁺ T lymphocytes, a lower frequency of effector/memory subsets (CCR7⁻CD45RA⁻ T cells among the total CD8⁺ T cells) and a higher frequency of severely exhausted populations (TIGIT⁺ T cells among PD-1⁺CD8⁺ T cells) were associated with HPD and inferior survival rate.ConclusionHPD is common in NSCLC patients treated with PD-1/PD-L1 inhibitors. Biomarkers derived from rationally designed analysis may successfully predict HPD and worse outcomes, meriting further investigation of HPD.     

The Proangiogenic Phenotype of Natural Killer Cells in Patients with Non-Small Cell Lung Cancer

The tumor microenvironment can polarize innate immune cells to a proangiogenic phenotype. Decidual natural killer (dNK) cells show an angiogenic phenotype, yet the role for NK innate lymphoid cells in tumor angiogenesis remains to be defined. We investigated NK cells from patients with surgically resected non-small cell lung cancer (NSCLC) and controls using flow cytometric and functional analyses. The CD56⁺CD16^- NK subset in NSCLC patients, which represents the predominant NK subset in tumors and a minor subset in adjacent lung and peripheral blood, was associated with vascular endothelial growth factor (VEGF), placental growth factor (PIGF), and interleukin-8 (IL-8)/CXCL8 production. Peripheral blood CD56⁺CD16^- NK cells from patients with the squamous cell carcinoma (SCC) subtype showed higher VEGF and PlGF production compared to those from patients with adenocarcinoma (AdC) and controls. Higher IL-8 production was found for both SCC and AdC compared to controls. Supernatants derived from NSCLC CD56⁺CD16^- NK cells induced endothelial cell chemotaxis and formation of capillary-like structures in vitro, particularly evident in SCC patients and absent from controls. Finally, exposure to transforming growth factor-β₁ (TGFβ₁), a cytokine associated with dNK polarization, upregulated VEGF and PlGF in peripheral blood CD56⁺CD16^- NK cells from healthy subjects. Our data suggest that NK cells in NSCLC act as proangiogenic cells, particularly evident for SCC and in part mediated by TGFβ₁.     

Spectral CT imaging as a new quantitative tool？ Assessment of perfusion defects of pulmonary parenchyma in patients with lung cancer

Objective： This study investigated the capability of dual-energy spectral computed tomography （CT） to quantitatively evaluate lung perfusion defects that are induced by central lung cancer. Methods： Thirty-two patients with central lung cancer underwent CT angiography using spectral imaging. A univariate general linear model was conducted to analyze the variance of iodine concentration/CT value with three factors of lung fields. A paired t-test was used to compare iodine concentrations and CT values between the distal end of lung cancer and the corresponding area in the contralateral normal lung. Results： Iodine concentrations increased progressively in the far, intermediate and near ground sides in the normal lung fields at 0.60±0.28, 0.93±0.27 and 1.25±0.38 mg/mL, respectively （P〈0.001）. The same trend was observed for the CT values [-（840.64±49.08）, -（812.66±50.85） and -（760.83±89.17） HU, P〈0.001]. The iodine concentration （0.70±0.42 mg/mL） of the lung field in the distal end of lung cancer was significantly lower than the corresponding area in the contralateral normal lung （1.19±0.62 mg/mL） （t=-7.23, P〈0.001）. However, the CT value of lung field in the distal end of lung cancer was significantly higher than the corresponding area in the contralateral normal lung [-（765.29±93.34） HU vs. -（800.07±76.18） HU, t=3.564, P=0.001]. Conclusions： Spectral CT imaging based on the spectral differentiation of iodine is feasible and can quantitatively evaluate pulmonary perfusion and identify perfusion defects that are induced by central lung cancer. Spectral CT seems to be a promising technique for the simultaneous evaluation of both morphological and functional lung information.     

Significance of combined detection of LunX mRNA and tumor markers in diagnosis of lung carcinoma简

Objective：To evaluate the significance of combined detection of LunX mRNA,carcinoembryonic antigen （CEA）,neuron-specific enolase （NSE）,and cytokeratin 21-1 fragment （CYFRA21-1） in clinical diagnosis of lung carcinoma.Methods：Based on the quantitative RT-PCR and chemiluminescence immunoassay,the expression levels of LunX mRNA,CEA,NSE,and CYFRA21-1 in 113 patients with lung carcinoma （case group） and 30 healthy participants （control group） were detected.Meantime,the sensitivity,specificity,and accuracy of the combination detection were also explored.Results：The positive rates of LunX mRNA in peripheral blood and CEA,NSE,and CYFRA21-1 in serum were significandy higher in case group than those in control group （x2=17.295,16.825,19.148,and 17.450; P＜0.05）.There was no statistical significance when positive rate of LunX mRNA was evaluated among different pathological types （x2=0.047,P＞0.05）.The positive rate of LunX mRNA in stage Ⅰ ＋ Ⅱ,Ⅲ,and Ⅳ had a significandy increasing tendency （x2=10.565,32.462,P＜0.05）.The positive rate of CYFRA21-1 was highest in squamous carcinoma （78.5％）,the positive rate of NSE was highest in small cell carcinoma （86.7％）,and the positive rate of CEA wag highest in lung adenocarcinoma （80.4％）.The sensitivity and accuracy of the combination detection were 91.1％ and 88.1％,respectively.Conclusions：The combined detection of LunX mRNA and tumor markers （TMs） including CEA,NSE,and CYFRA21-1 in peripheral blood is helpful to increase the diagnostic accuracy of lmg cancer.Also,it can inform the pathological typing of lung carcinoma.