Tumor-infiltrating Mononuclear Cells Research Articles

Does CSF1R Blockade Turn into Friendly Fire?

In this issue of Cancer Cell, Kumar etal. describe how CSF1R blockade induces not only an expected deprivation of tumor-associated macrophages, but also an accumulation of tumor-infiltrating polymorphonuclear mononuclear cells caused by Cxcl-1 released from cancer-associated fibroblasts.

Abstract 3995: Identification of receptor tyrosine kinases (RTKs) expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMC)

Abstract Aim of this study is to discover expression levels of RTKs (VEGFR1, VEGFR2, PDGFRa, PDGFRb) in CD45+ population of PMBC and TILs isolated from patients with clear cell renal cell cancer (RCC). Here we present preliminary data. Species of tumors and blood were prospective collected from 9 patients with RCC after radical nephrectomy or tumor resection from July to October 2016. TILs isolation was performed under standard protocol with slight modification. Briefly, tumor was shredded with scissors on small pieces of 2-3 mm and twice washed in RPMI 1640 media by centrifugation for 10 min at 1500 rpm. After centrifugation, pellet was diluted RPMI 1640 with enzyme mix (Collagenase and Dispase II) on Petri dish and placed on shaker-thermostat andincubate sample for 12h at 37C with continuous shaking. After incubation, cell suspension was passed thought a cell strainer, mesh size 70 μm, placed on a 50 mL tube and washed twice washed in RPMI 1640 media by centrifugation. For TILs isolation cell suspension was placed on Ficoll-Paque Plus and isolated by centrifugation for 25 min at 1500 rpm. Cell pellet was resuspended in RPMI 1640 media. PMBC was isolated from whole blood using the Ficoll-Paque method. Isolated TIL and PMBC were prepared for flow cytometry at typical protocol with or without permeabilization. Cells were stained with anti-CD45 FITC-conjugated antibodies (Novus), primary anti-VEGFR1, anti-VEGFR2, anti-PDGFRa, anti-PDGFRb (Abcam) mouse antibodies and Alexa-568 conjuated secondary anti-mouse antibodies (Invitrogen) and analyzed on BD Canto II flow cytometer. Expression of RTKs on CD45+ cell population were evaluated. Among PMBC 86±9% of cells was CD45-positive and expressed RTKs (10±1% cells were positive for VEGFR1 and VEGFR2, and 6±2% cells were positive for PDGRFa and PDGFRb). TILs contained 65±18% of CD45-positive cells and did not express RTKs on the cell surface (less than 1% of cells), but intracellular staining showed presence of RTKs: 8±2% of cells were VEGFR1 or VEGFR2-positive, 20±8% of cells were PDGFRa or PDGFRb-positive. Preliminary data demonstrate extracellular expression of RTKs on PBMC and intracellular expression of RTKs in TILs. The study will be continued. Citation Format: Dmitry Khochenkov, Maria Volkova, Ilya Tsimafeyeu. Identification of receptor tyrosine kinases (RTKs) expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3995. doi:10.1158/1538-7445.AM2017-3995

Prognostic value of programmed death-ligand 1 expression in patients with stage III colorectal cancer.

The programmed death‐1/programmed death‐ligand 1 (PD‐L1) pathway is a negative feedback pathway that suppresses the activity of T cells. Previous studies reported that high PD‐L1 expression on tumor cells (TC) was associated with poor survival in patients with colorectal cancer; however, the prognostic evaluation of these studies was limited because they included patients at various disease stages. The purpose of the present study was to evaluate the relationship between PD‐L1 status in the immune microenvironment and the clinicopathological features of stage III colorectal cancer. Two hundred and thirty‐five patients were included in the analysis. PD‐L1 expression on TC and tumor‐infiltrating mononuclear cells (TIMC) was evaluated by immunohistochemistry. The median follow‐up of thisi study was 52.9 months. A total of 8.1% of stage III colorectal cancer showed high PD‐L1 expression on TC and 15.3% showed high PD‐L1 expression on TIMC. Patients with high PD‐L1 expression on TC had significantly shorter disease‐free survival (DFS) than patients with low expression (hazard ratio [HR] 2.36; 95% confidence interval [CI], 1.21–4.62; P = 0.012). In addition, patients with high PD‐L1 expression on TIMC were associated with longer DFS than patients with low expression (HR 0.40; 95% CI, 0.16–0.98; P = 0.046). These findings suggest that PD‐L1 expression status may be a new predictor of recurrence for stage III colorectal cancer patients and highlight the necessity of evaluating PD‐L1 expression on TC and TIMC separately in the tumor microenvironment.

Prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating mononuclear cells in upper tract urothelial carcinoma.

Immunotherapy targeting the programmed death-1 (PD-1) receptor/PD-1 ligand (PD-L1) pathway has shown promising results in several malignancies. However, the prognostic significance of PD-L1 expression remains unknown in patients with upper tract urothelial carcinoma (UTUC). This study aimed to evaluate PD-L1 expression and its association with clinicopathological characteristics and oncological outcomes in UTUC patients. PD-L1 expression on tumor cells and tumor-infiltrating mononuclear cells (TIMCs), and E-cadherin and N-cadherin expression on tumor cells were assessed by immunohistochemistry in a cohort of 162 patients with UTUC. Associations of PD-L1 expression on tumor cells and TIMCs with clinicopathological characteristics and cancer-specific survival (CSS) were evaluated. Out of 162 patients, 20 (12.3%) and 35 (21.6%) had positive PD-L1 expression on tumor cells and TIMCs, respectively. Decreased E-cadherin expression was associated with PD-L1 positivity on tumor cells (P=0.048) and PD-L1 negativity on TIMCs (P=0.033). PD-L1 expression on tumor cells was higher in patients with preoperative chronic kidney disease (CKD) stage 4-5 than in those with no CKD or CKD stage 1-3 (P=0.011). PD-L1 was differentially expressed in tumor cells and TIMCs in UTUC. Multivariate analyses revealed that PD-L1 expression on tumor cells independently predicted shorter CSS (P=0.012), whereas PD-L1 expression on TIMCs independently predicted longer CSS (P=0.034).

Abstract IA17: Technologies for personalizing cancer immunotherapies

Abstract At the heart of most cancer immunotherapies are the specific interactions between tumor-infiltrating T cells, and the antigens that attract those T cells into the tumor. It has become increasingly apparent that (mutation containing) neoantigens likely play an important role in this regard. Putative neoantigens are identified by tumor exome analysis, but finding which of those candidates actually promote T cell tumor infiltration is challenging, as is pairing such antigens with the cognate T cell receptor (TCR) genes. Technologies that can address this challenge can help pave the way for personalized immunotherapies. For example, the active neoantigens can be engineered into personalized cell-based cancer vaccines, while the cognate TCRs represent engineered T cell therapy opportunities. I will discuss a nanotech/microchip tool that we have recently developed to address this challenge. The platform allows for the non-destructive enumeration of neoantigen specific T cell populations from either tumor infiltrating lymphocytes (TILs) or unexpanded peripheral blood mononuclear cells (PBMCs), beginning with as few as 10,000 CD8+ cells. I will discuss the application of the platform for kinetic studies of patient responses to checkpoint inhibitor therapy (via blood analysis), as well as applications for matching neoantigen-specificity with T cell receptor genes. I will also discuss how the resultant data can be used to guide the refinement of neoantigen prediction algorithms. Citation Format: Songming Peng, Jesse Zaretsky, Michael Bethune, Alice Hsu, John E. Heath, Won Jun Noh, Shannon Esswein, Antoni Ribas, David Baltimore, James R. Heath. Technologies for personalizing cancer immunotherapies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA17.

PD-1/PD-L1 expression in chromophobe renal cell carcinoma: An immunological exception?

Immune checkpoint inhibitors targeting the inhibitory cross talk between tumor and immune cells have been approved for therapy in renal cell carcinoma (RCC). In contrast to clear cell RCC, little is known on PD-1/PD-L1 expression patterns in rarer RCC subtypes. The aim of this study was to evaluate the prevalence, distribution and prognostic impact of PD-1 and PD-L1 expression in chromophobe (ch)RCC. Patients who underwent renal surgery due to chRCC were retrospectively evaluated. Tumor specimen was analyzed for PD-1 and PD-L1 expression by immunohistochemistry. Expression data were correlated with clinic-pathological parameters including patient survival. Eighty-one chRCC patients were eligible for analysis, thereof 25 (30.9%) and 11 (13.6%) patients were positive for PD-1+ tumor-infiltrating mononuclear cells (TIMCs) and tumoral PD-L1+ expression, respectively. No significant associations were found for PD-1+ TIMC or tumoral PD-L1+ expression and clinical attributes. In addition, no differences in 5- and 10-year overall survival for PD-1- TIMC compared to PD-1+ TIMC (90.5 and 72.2 vs. 100 and 75%; p=0.41) and for PD-L1- tumors compared to PD-L1+ tumors (91.9 and 76.4 vs. 100 and 50%; p=0.48) were observed. In conclusion, to our knowledge this is the first study to evaluate the prognostic impact of PD-1 and PD-L1 in chRCC. PD-L1 does seem to be expressed in a minority of all chRCC, likewise only a minority of chRCC was infiltrated by PD-1-positive inflammatory cells. Neither PD-1+ TIMC nor tumoral PD-L1+ expression was associated with parameters of aggressiveness or survival.

Correlation of Apobec Mrna Expression with overall Survival and pd-l1 Expression in Urothelial Carcinoma.

Metastatic urothelial carcinoma (mUC) has a very high mutational rate and is associated with an APOBEC mutation signature. We examined the correlation of APOBEC expression with overall survival (OS) and PD-L1 expression in a cohort of 73 mUC patients. mRNA expression of APOBEC3 family of genes (A3A, A3B, A3C, A3F_a, A3F_b, A3G, A3H) was measured using Nanostring. PD-L1 expression, evaluated by immunohistochemistry, on tumor infiltrating mononuclear cells (TIMCs) and tumor cells was scored from 0 to 4, with 2–4 being positive. Wilcoxon’s non-parametric tests assessed the association of APOBEC and PD-L1. The Cox regression model assessed the association of APOBEC with OS. All APOBEC genes were expressed in mUC. Increased A3A, A3D, and A3H expression associates with PD-L1 positive TIMCs (p = 0.0009, 0.009, 0.06). Decreased A3B expression was marginally associated with PD-L1 positive TIMCs expression (p = 0.05). Increased A3F_a and A3F_b expression was associated with increased expression of PD-L1 on tumor cells (p = 0.05). Increased expression of A3D and A3H was associated with longer OS (p = 0.0009). Specific APOBEC genes have different effects on mUC in terms of survival and PD-L1 expression. A3D and A3H may have the most important role in mUC as they are associated with OS and PD-L1 TIMC expression.

Do programmed death 1 (PD-1) and its ligand (PD-L1) play a role in patients with non-clear cell renal cell carcinoma?

Clinical trials targeting programmed death 1 (PD-1) and its ligand PD-L1 (PD-L1) for metastatic renal cell cancer (RCC) are ongoing. The aim of this study is to validate their roles as prognostic markers in non-clear cell (non-cc) RCC. Sixty-four non-cc RCC tissue specimens were collected from patients undergoing renal tumor surgery. Expressions of biomarkers were assessed using immunohistochemistry and compared with clinical characteristics. Survival analyses were performed with a median follow-up of 77.5 (range: 0-176) months. No significant correlations were found for PD-1(+) tumor-infiltrating mononuclear cells (TIMC) or PD-L1(+) expression and clinical attributes in patients with non-cc RCC. Kaplan-Meier analysis revealed no differences in 5- and 10-year cancer-specific survival (CSS) for PD-1(-) TIMC compared to PD-1(+) TIMC (71.4 and 63% versus 72.2 and 61.9%; p=0.88). Intratumoral expression of PD-L1 did not appear to influence the 5- and 10-year CSS significantly, even though a trend was identified (68 and 53.6% versus 80.1 and 75.7%; p=0.08). In multivariate analysis, neither PD-1(+) TIMC nor intratumoral PD-L1(+) expression proved to be independent predictors of CSS (p=0.99 and p=0.68, respectively). Our study demonstrates that PD-1(+) TIMC and intratumoral PD-L1(+) expression did not significantly impact tumor aggressiveness or clinical outcome in non-ccRCC specimens. Due to rare incidence of non-cc RCC in particular according to PD-L1 expression, further analyzes are warranted.

AB243. Deep sequencing reveals intensive interindividual and intraindividual heterogeneity in TCR-beta repertoire across multiple renal cell carcinoma subtypes

BackgroundTumor immunogenicity has been proved to have an essential role in tumor development and metastasis. Cancer cell recognition of T cells can be potentially utilized in tumor prognosis and immunotherapy. However, the understanding of immune responses and T cell receptor (TCR) repertoires in many types of tumor is yet to be complete. Our aim was to explore the TCR beta-chain (TCRb) heterogeneity across renal cell carcinoma (RCC) patients, as well as the TCRb heterogeneity between tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs).MethodsTumor and blood samples of thirty-nine treatment-naïve RCC patients, and blood samples of two renal neoplasm patients and ten healthy volunteers were obtained. Genomic DNA was extracted from the frozen tumor tissues and the isolated PBMCs. To prepare the TCRb library, primers were designed to amplify the CDR3 region of the TCRb gene for the followed high-throughput sequencing. The sequencing data was analyzed by an in-house immune repertoire analyzing pipeline, including Shannon diversity, clonotype abundance, pair-wise overlap and distance, and other analyses.ResultsTCRb repertoires in the RCC tumor tissues have lower diversity compared to the RCC PBMC samples while the TCRb repertoires in the non-carcinoma PBMC samples possess the highest diversity among them. The abundance of highly expressed clonotypes (HECs) in the RCC tumor samples is higher than the HECs in the RCC PBMC samples, whereas the abundance of HECs in the non-carcinoma PBMC samples was the lowest. The pair-wise distance data generated by comparing the overlap of any two included samples suggest that TCRb repertoires in RCC do not produce distinguishable pattern across pathologically classified subtypes. The comparison of two cladograms generated from the pair-wise distances with or without the HECs suggests that HEC was a major contributor to the intraindividual difference in TCRb repertoires between TILs and PBMCs.ConclusionsIntensive interindividual and intraindividual heterogeneity in TCRb repertoire across multiple RCC subtypes can be observed in the aspect of diversity, abundance of HECs, and pair-wise distance; the intraindividual heterogeneity in RCC is mainly contributed by HECs.

Mismatch repair gene defects in sporadic colorectal cancer enhance immune surveillance.

There is evidence that colorectal cancers (CRC) with DNA mismatch repair deficiency (MMR-D) are associated with a better prognosis than the generality of large bowel malignancies. Since an active immune surveillance process has been demonstrated to influence CRC outcome, we investigated whether MMR-D can enhance the immune response in CRC. A group of 113 consecutive patients operated for CRC (42 stage I or II and 71 with stage III or IV) was retrospectively analyzed. The expression of MMR genes (MSH2, MLH1, MSH6 and PSM2) and co-stimulatory molecule CD80 was assessed by tissue microarray immunohistochemistry. In addition, tumor infiltrating mononuclear cells (TIMC) and T cell subpopulations (CD4, CD8, T-bet and FoxP-3) were quantified. The effect of specific siRNA (siMSH2, siMLH1, siMSH6 and siPSM2) transfection in HT29 on CD80 expression was quantified by flow cytometry. Non parametric statistics and survival analysis were used. Patients with MMR-D showed a higher T-bet/CD4 ratio (p = 0.02), a higher rate of CD80 expression and CD8 lymphocyte infiltration compared to those with no MMR-D. Moreover, in the MMR-D group, the Treg marker FoxP-3 was not expressed (p = 0.05). MMR-D patients with stage I or II and T-bet expression had a significant better survival (p = 0.009). Silencing of MSH2, MLH1 and MSH6, but not PSM2, significantly increased the rate of CD80+ HT29 cells (p = 0.007, p = 0.023 and p = 0.015, respectively). CRC with MMR-D showed a higher CD80 expression, and CD8+ and Th1 T-cell infiltration. In vitro silencing of MSH2, MLH1 and MSH6 significantly increased CD80+ cell rate. These results suggest an enhanced immune surveillance mechanism in presence of MMR-D.

Differential Expression of PD-L1 between Primary and Metastatic Sites in Clear-Cell Renal Cell Carcinoma.

PD-L1 expression in primary clear-cell renal cell carcinoma (ccRCC) increases the likelihood of response to anti-PD-1 inhibition, but fails to identify all responders. We hypothesized that PD-L1 levels assessed in randomly selected areas of the primary tumors may not accurately reflect expression levels in metastatic lesions, which are the target of systemic therapy. Therefore, we compared PD-L1 expression in a series of primary ccRCC and their metastases. Tissue blocks from 53 primary ccRCCs and 76 corresponding metastases were retrieved. Areas with predominant and highest nuclear grade were selected. Slides were immunostained with a validated anti-PD-L1 antibody (405.9A11). Membranous expression in tumor cells was quantified using H-score. Expression in tumor-infiltrating mononuclear cells (TIMC) was quantified using a combined score. Discordant tumor cell PD-L1 staining between primary tumors and metastases was observed in 11 of 53 cases (20.8%). Overall, tumor cell PD-L1 levels were not different in primary tumors and metastases (P = 0.51). Tumor cell PD-L1 positivity was associated with higher T stage (P = 0.03) and higher Fuhrman nuclear grade (P < 0.01). Within individual lesions, PD-L1 positivity was heterogeneous and almost exclusively detected in high nuclear grade areas (P < 0.001). No difference was found in PD-L1 levels in TIMCs between primary tumors and metastases (P = 0.82). The heterogeneity of PD-L1 expression in ccRCC suggests that its assessment as a predictive biomarker for PD-1 blockade may require analysis of metastatic lesions. Notably, because PD-L1 expression was mostly detected in high nuclear grade areas, to avoid false-negative results, these areas should be specifically selected for assessment.

The status of PD-L1 and tumor-infiltrating immune cells predict resistance and poor prognosis in BRAFi-treated melanoma patients harboring mutant BRAFV600

BRAF inhibitors (BRAFi) improve survival in metastatic melanoma patients (MMP) but the duration of clinical benefit is limited by development of drug resistance. Here, we investigated whether the expression of programmed death-ligand 1 (PD-L1) and the density of tumor-infiltrating mononuclear cells (TIMC) predict the occurrence of resistance, hence affecting the clinical outcome in BRAFi-treated MMP. PD-L1 expression (cutoff 5%) was analyzed by immunohistochemistry with two different antibodies in BRAF(V600)-mutated formalin-fixed and paraffin-embedded samples from 80 consecutive MMP treated with BRAFi at a single institution. TIMC were evaluated by conventional hematoxylin and eosin staining. Forty-six and 34 patients received vemurafenib and dabrafenib, respectively. Membranous expression of PD-L1 was detected in 28/80 (35%) of patients. At multivariate analysis, absence of tumoral PD-L1 staining [odd ratio (OR) 10.8, 95% confidence interval (CI) 2.7-43.3, P < 0.001] and the presence of TIMC (OR 6.5, 95% CI 1.7-24.3, P < 0.005) were associated with a better response to treatment. Median progression-free survival (PFS) and overall survival were 10 and 15 months, respectively. By multivariate assessment, PD-L1 expression [hazard ratio (HR) 4.3, 95% CI 2.1-8.7, P < 0.0001] and absence of TIMC (HR 2.5, 95% CI 1.4-4.7, P < 0.002) correlated with shorter PFS. PD-L1 overexpression (HR 6.2, 95% CI 2.8-14.2, P < 0.0001) and absence of TIMC (HR 3.1, 95% CI 1.5-6.5, P < 0.002) were independent prognostic factors for melanoma-specific survival. Our results provide the first proof-of-principle evidence for the predictive and prognostic relevance of PD-L1 immunohistochemical expression and density of immune cell infiltration in BRAF(V600)-mutated MMP treated with BRAFi.

2647 Prognostic significance of PD-L1 expression on tumor-infiltrating mononuclear cells in patients with urothelial carcinoma

2647 Prognostic significance of PD-L1 expression on tumor-infiltrating mononuclear cells in patients with urothelial carcinoma

Association of PD-L1 expression on tumor-infiltrating mononuclear cells and overall survival in patients with urothelial carcinoma

Programmed death-1 (PD-1) receptor/PD-1 ligand (PD-L1) pathway negatively regulates T-cell-mediated responses. The prognostic impact of PD-L1 expression needs to be defined in urothelial carcinoma (UC). Formalin-fixed paraffin-embedded tumor samples from 160 patients with UC were retrieved. PD-L1 expression was evaluated by immunohistochemistry using a mouse monoclonal anti-PD-L1 antibody (405.9A11). PD-L1 positivity on tumor cell membrane was defined as ≥5% of tumor cell membrane staining. The extent of tumor-infiltrating mononuclear cells (TIMCs) as well as PD-L1 expression on TIMCs was scored from 0 to 4. A score of 2, 3, or 4 was considered PD-L1-positive. Clinico-pathological variables were documented. The Cox regression model was used to assess the association of PD-L1 expression with overall survival (OS) in patients who developed metastases. TIMCs were present in 143 of the 160 patient samples. Out of 160 samples, 32 (20%) had positive PD-L1 expression in tumor cell membrane. Out of 143 samples with TIMCs, 58 (40%) had positive PD-L1 expression in TIMCs. Smoking history, prior BCG use and chromosome 9 loss did not correlate with PD-L1 expression in either tumor cell membrane or TIMCs. PD-L1 positivity was not different between non-invasive or invasive UC. In patients who developed metastases (M1) and were treated with systemic therapy (n = 100), PD-L1 positivity on tumor cell membrane was seen in 14% of patients and did not correlate with OS (P = 0.45). Out of 89 M1 patients who had evaluable PD-L1 on TIMCs, PD-L1 expression was seen in 33% of patients and was significantly associated with longer OS on multivariate analysis (P = 0.0007). PD-L1 is widely expressed in tumor cell membrane and TIMCs in UC. PD-L1 in tumor cells was not predictive of OS. However, positive PD-L1 expression in TIMCs was significantly associated with longer survival in those patients who developed metastases.

Immunoexpression of metalloproteinase 14 and tissue inhibitor of metalloproteinase 2 in colorectal carcinomas and lymph node metastases

Changes in metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) have been associated with tumor progression in colorectal cancer. However, the role of MMP-14 and TIMP-2 has yet to be determined. We investigated the differential expression of MMP-14 and TIMP-2 in colorectal carcinomas of the left and the right colon, as well as in mononuclear cells in primary tumors and their lymph node metastases. We performed an immunohistochemistry analysis of tumor samples obtained from 50 cases of colorectal cancer. We found that MMP-14 staining was positive in 100 % of cases, in contrast to normal mucosa (86 % positivity, P = 0.0451). Additionally, neoplastic cells showed a higher frequency of TIMP-2-positive staining (70 % versus 14 % of normal mucosa, P = 0.0004). Furthermore, MMP-14 expression in primary tumor-associated mononuclear cells was higher in cases without lymph node metastases (N0) in comparison to more advanced carcinomas (N1–N3) (P = 0.0353). MMP-14 and TIMP-2 expression was observed in neoplastic cells in primary tumors, with a higher frequency of increased expression of MMP-14 (82 %) than increased expression of TIMP-2 (22 %, P < 0.0001). The expression of MMP-14 and TIMP-2 was evaluated in each cell type and at each site, and the frequency of TIMP-2 expression in colonic lesions and in the lymph nodes was significantly higher than in tumor-infiltrating mononuclear cells (P = 0.0003 and P = 0.0406, respectively). Expression of MMP-14 and TIMP-2 in primary colorectal carcinomas and in their lymph node metastases suggests the involvement of these proteins in local invasion and tumor progression.

Programmed death ligand-1 expression in adrenocortical carcinoma: an exploratory biomarker study.

BackgroundAdrenocortical carcinoma (ACC) is a rare tumor in which prognostic factors are still not well established. Programmed Death Ligand-1 (PD-L1) expression in ACC and its association with clinico-pathological features and survival outcomes are unknown.MethodsFormalin-fixed paraffin-embedded (FFPE) specimens were obtained from 28 patients with ACC. PD-L1 expression was evaluated by immunohistochemistry (IHC) in both tumor cell membrane and tumor infiltrating mononuclear cells (TIMC). PD-L1 positivity on tumor cells was defined as ≥5% tumor cell membrane staining. TIMC were evaluated by IHC using a CD45 monoclonal antibody. For PD-L1 expression in TIMC, a combined score based on the extent of infiltrates and percentage of positive cells was developed. Any score greater that zero was considered PD-L1 positive. Baseline clinico-pathological characteristics and follow up data were retrospectively collected. Comparisons between PD-L1 expression and clinico-pathological features were evaluated using unpaired t-test and Fisher’s exact test. Kaplan-Meier method and log-rank test were used to assess association between PD-L1 expression and 5-year overall survival (OS).ResultsAmong 28 patients with surgically treated ACC, 3 (10.7%) were considered PD-L1 positive on tumor cell membrane. On the other hand, PD-L1 expression in TIMC was performed in 27 specimens and PD-L1 positive staining was observed in 19 (70.4%) patients. PD-L1 positivity in either tumor cell membrane or TIMC was not significantly associated with higher stage at diagnosis, higher tumor grade, excessive hormone secretion, or OS.ConclusionsPD-L1 expression can exist in ACC in both tumor cell membrane and TIMC with no relationship to clinico-pathologic parameters or survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0047-3) contains supplementary material, which is available to authorized users.

PD-L1 expression in nonclear-cell renal cell carcinoma

Programmed death ligand-1 (PD-L1) expression in nonclear-cell RCC (non-ccRCC) and its association with clinical outcomes are unknown. Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 101 patients with non-ccRCC. PD-L1 expression was evaluated by immunohistochemistry in both tumor cell membrane and tumor-infiltrating mononuclear cells (TIMC). PD-L1 tumor positivity was defined as ≥5% tumor cell membrane staining. For PD-L1 expression in TIMC, a combined score based on the extent of infiltrate and percentage of positive cells was used. Baseline clinico-pathological characteristics and outcome data [time to recurrence (TTR) and overall survival (OS)] were correlated with PD-L1 staining. Among 101 patients, 11 (10.9%) were considered PD-L1+ in tumor cells: 2/36 (5.6%) of chromophobe RCC, 5/50 (10%) of papillary RCC, 3/10 (30%) of Xp11.2 translocation RCC and 1/5 (20%) of collecting duct carcinoma. PD-L1 positivity (PD-L1+) in tumor cells was significantly associated with higher stage (P = 0.01) and grade (P = 0.03), as well as shorter OS (P < 0.001). On the other hand, PD-L1 positivity by TIMC was observed in 57 (56.4%) patients: 13/36 (36.1%) of chromophobe RCC, 30/50 (60%) of papillary RCC, 9/10 (90%) of Xp11.2 translocation RCC and 5/5 (100%) of collecting duct carcinoma. A trend toward shorter OS was observed in patients with PD-L1+ in TIMC (P = 0.08). PD-L1+ in both tumor cell membrane and TIMC cells were associated with shorter TTR (P = 0.02 and P = 0.03, respectively). In non-ccRCC, patients with PD-L1+ tumors appear to have worse clinical outcomes, although only PD-L1 positivity in tumor cells is associated with higher tumor stage and grade.

Abstract 187: Absence of S100A9 confers survival advantage in an aggressive de novo mouse model of glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most common (60%) primary brain tumor in adults and constitutes a major challenge for both patients and clinicians, with a median survival of only 15-21 months when treated with surgery, radiation therapy and temozolomide. To improve patient outcome, a promising and safe avenue of adjuvant treatment is immune therapy with dendritic cell vaccines or adoptive T cell transfer. These treatments are however hindered by the immune suppressive environment induced by GBMs. Understanding the mechanisms by which GBM suppress the anti-tumor immune response is paramount in order to develop successful immune therapies. The Sleeping Beauty transposase system was used to modify the genetic makeup of stem cells in the sub-ventricular zone of neonatal mice and thus induce de novo glioblastomas with several combinations of genes. The most aggressive tumors where induced when combining NRAS and SV-40 Large T antigen (NLgT) generating invasive tumors which show the histological hallmarks human GBM (WHO grade IV) with pseudo-pallisading necrosis, neovascularization and hemorrhages, rendering the mice moribund with a median survival of 30 days. Myeloid derived suppressive cells, a heterogeneous population of immature bone marrow derived cells, are induced by cancers and other consumptive diseases and strongly inhibit adaptive and innate immune responses. We show that MDSCs isolated from de novo GBMs, inhibit antigen-specific and antigen non-specific T cell proliferation. Tumor infiltrating MDSCs highly express the pro-inflammatory secreted calcium binding protein S100A9 and its cognate receptors RAGE and TLR4. In bone marrow cultures, conditioned media from primary cell lines derived from NLgT tumors induce a marked expansion (60-70%) of myeloid derived suppressor cells (MDSCs) and enhance the expression of S100A9. Mice deficient for S100A9 with de novo NLgT tumors show an increased survival compared to wild-type animals (median survival= 47days, a 56% improvement). Analysis of tumor-infiltrating mononuclear cells in moribund NLgT wild-type and S100A9KO mice shows no significant difference in percent infiltrating MDSCs, macrophages and dendritic cells (% of CD45+), however the percent of CD8+ cytotoxic T lymphocytes is increased in S100A9 KO (16% vs. 6% p=0.0228). In addition, expression of MHCII is increased in macrophages and dendritic cells from S100A9 KO mice when compared to wild type mice. Taken together these data suggest that the absence of S100A9 confers a survival advantage by allowing a stronger anti-tumor immune response with:(1) increased number of cytotoxic T lymphocytes and (2) increased maturation of antigen presenting cells. Experiments are currently underway to identify the detailed cellular and molecular events, which give rise to this phenotype. This study was supported by NIH/RO1 NS057711 and NS074387. Citation Format: Alexandra Calinescu, Hikmat Assi, Bradley Kolb, Carl Koschmann, Pedro R. Lowenstein, John Ohlfest, Maria G. Castro. Absence of S100A9 confers survival advantage in an aggressive de novo mouse model of glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 187. doi:10.1158/1538-7445.AM2014-187

Purification of Tumor-Associated Macrophages (TAM) and Tumor-Associated Dendritic Cells (TADC)

Tumors are heterogeneous microenvironments where complex interactions take place between neoplastic cells and infiltrating inflammatory cells, such as tumor-associated macrophages (TAM) and tumor-associated dendritic cells (TADC). The relevance of tumor-infiltrating mononuclear myeloid cells is underscored by clinical studies showing a correlation between their abundance and poor prognosis (Laoui et al., 2011). These cells are able to promote tumor progression via several mechanisms, including induction of angiogenesis, remodeling of the extracellular matrix, stimulation of cancer cell proliferation and metastasis and the inhibition of adaptive immunity (Laoui et al., 2011). Moreover, mononuclear myeloid cells are characterized by plasticity and versatility in response to microenvironmental signals, resulting in different activation states, as illustrated by the presence of distinct functional TAM subsets in tumors (Movahedi et al., 2010; Laoui et al., 2014). Here, we describe a valuable isolation technique for TAM and TADC permitting their molecular and functional characterization., 肿瘤是异质微环境，其中在肿瘤细胞和浸润性炎症细胞例如肿瘤相关巨噬细胞（TAM）和肿瘤相关树突细胞（TADC）之间发生复杂的相互作用。临床研究强调了肿瘤浸润性单核骨髓细胞的相关性，显示其丰度与不良预后之间的相关性（Laou等人，2011）。这些细胞能够通过几种机制促进肿瘤进展，包括诱导血管生成，重塑细胞外基质，刺激癌细胞增殖和转移以及抑制适应性免疫（Laoui等人，2011 ）。此外，单核骨髓细胞的特征在于响应于微环境信号的可塑性和通用性，导致不同的活化状态，如通过在肿瘤中存在不同的功能性TAM亚型所说明的（Movahedi等人，2010; Laoui et al。，2014）。在这里，我们描述了允许其分子和功能表征的TAM和TADC的有价值的分离技术。

Expression of TLR9 in tumor‐infiltrating mononuclear cells enhances angiogenesis and is associated with a worse survival in lung cancer

Toll-like receptors (TLRs) play a crucial role in the innate and adaptive immune responses against microbial infection, tissue injury and cancer. Ligands of TLR9 have been developed as therapy in non-small-cell lung carcinoma (NSCLC). However, phase III clinical trials in metastatic NSCLC were negative. Our objective was to determine whether TLR9 affects tumor growth. We generated a mouse model of lung adenocarcinoma (ADC) mutated for K-ras (K-ras(LA1) ), with and without TLR9 inactivation (K-ras(LA1) TLR9(-/-) and K-ras(LA1) TLR9(+/+) , respectively). TLR9 was functionally expressed only in mononuclear cells of K-ras(LA1) TLR9(+/+) mice. These mice had significantly worse survival and a higher tumor burden than K-ras(LA1) TLR9(-/-) mice. Lung tumors were analyzed for 24 cytokines/growth factors using Bio-Plex multiplex bead-based assays. Factor VIII was assessed by immunochemistry. Tumors from K-ras(LA1) TLR9(+/+) mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K-ras(LA1) TLR9(-/-) mice. LKR13 cells, an ADC cell line derived from K-ras(LA1) mice, were subcutaneously injected into TLR9(-/-) and TLR9(+/+) mice. Syngeneic tumors regressed in TLR9(-/-) but not in TLR9(+/+) mice. Peripheral blood mononuclear cells from TLR9(-/-) mice released less VEGF than those from TLR9(+/+) mice. In 61 patients with early-stage NSCLC, TLR9 was expressed in mononuclear cells that infiltrated tumors, as assessed by immunochemistry, and contributed to worse survival. Our results suggest that TLR9 expression in mononuclear cells was associated with an angiogenic phenotype and promoted lung cancer progression. These findings may aid clinical development of TLR9 ligands to treat cancers.