Specific immune landscapes and immune checkpoint expressions in histotypes and molecular subtypes of sarcoma.

Soft tissue sarcomas are a group of rare and aggressive connective tissue neoplasms for which curative therapeutic opportunities are limited in advanced phase. Clinical trials assessing immunotherapy in these tumors have so far reported limited efficacy. The objective of this study is to provide a description of the immunologic landscape of sarcomas to guide the next clinical trials of immunotherapy in these diseases. The gene expression profile of 93 immune checkpoint (ICP) and membrane markers (MM) of immune cells was analyzed in a series of 253 soft tissue sarcoma (synovial sarcoma, myxoid liposarcoma, sarcoma with complex genomic and GIST) using Agilent Whole Human Genome Microarrays. The unsupervised hierarchical clustering of gene expression level was found able to properly group patients according to the histological subgroup of sarcoma, indicating that each sarcoma subgroup is associated with a specific immune signature defined by its gene expression pattern. Using the prognostic impact of CIBERSORT signature on metastatic-free survival in each subgroup, specific target could be proposed for each of the four groups: Treg through ICOS and GITR in GIST, M0 macrophages in all four sarcoma subtypes, OX40 in SS, CD40 in GIST and SS. The immune landscape of sarcoma was found to be as heterogeneous as the histotypes and molecular subtypes, but strongly correlated to the histotype. Histotype adapted immunotherapeutic approaches in each sarcoma subtypes must be considered in view of these results, consistently with the already reported specific response of histotypes of ICPs.

The immune microenvironment of HPV-negative oral squamous cell carcinoma from never-smokers and never-drinkers patients suggests higher clinical benefit of IDO1 and PD1/PD-L1 blockade.

BACKGROUND: Never-smokers and never-drinkers patients (NSND) suffering from oral squamous cell carcinoma (OSCC) are epidemiologically different from smokers drinkers (SD). We therefore hypothesized that they harbored distinct targetable molecular alterations. PATIENTS AND METHODS: Data from The Cancer Genome Atlas (TCGA) (discovery set), Gene Expression Omnibus and Centre Léon Bérard (CLB) (three validation sets) with available gene expression profiles of HPV-negative OSCC from NSND and SD were mined. Protein expression profiles and genomic alterations were also analyzed from TCGA, and a functional pathway enrichment analysis was carried out. Formalin-fixed paraffin-embedded samples from 44 OSCC including 20 NSND and 24 SD treated at CLB were retrospectively collected to perform targeted-sequencing of 2559 transcripts (HTG EdgeSeq system), and CD3, CD4, CD8, IDO1, and PD-L1 expression analyses by immunohistochemistry (IHC). Enrichment of a six-gene interferon-γ signature of clinical response to pembrozulimab (PD-1 inhibitor) was evaluated in each sample from all cohorts, using the single sample gene set enrichment analysis method. RESULTS: A total of 854 genes and 29 proteins were found to be differentially expressed between NSND and SD in TCGA. Functional pathway analysis highlighted an overall enrichment for immune-related pathways in OSCC from NSND, especially involving T-cell activation. Interferon-γ response and PD1 signaling were strongly enriched in NSND. IDO1 and PD-L1 were overexpressed and the score of response to pembrolizumab was higher in NSND than in SD, although the mutational load was lower in NSND. IHC analyses in the CLB cohort evidenced IDO1 and PD-L1 overexpression in tumor cells that was associated with a higher rate of tumor-infiltrating T-cells in NSND compared with SD. CONCLUSION: The main biological and actionable difference between OSCC from NSND and SD lies in the immune microenvironment, suggesting a higher clinical benefit of PD-L1 and IDO1 inhibition in OSCC from NSND.

Pattern recognition receptors: immune targets to enhance cancer immunotherapy.

Durable tumor responses and significant levels of disease control rates have been described in more than 20 advanced/metastatic cancer types with B7-family immune checkpoint-targeted anti-CTLA-4, anti-PD-1, and anti-PD-L1 monoclonal antibodies. These results and the recent approvals of ipilimumab, pembrolizumab, nivolumab and atezolizumab are currently revolutionizing the way we envision the future of cancer care. However these clinical benefits are not observed in all cancer types and in every patient. Therefore, our clinical challenge is to identify therapeutic strategies which could overcome the primary and secondary resistances to these novel cancer immunotherapies. Pattern recognition receptors (PRRs) are other critical costimulatory molecules of immune cells, notably myeloid cells (macrophages and dendritic cells). They were initially described as sensors for 'danger signals' released by pathogens (e.g. viral DNA and bacterial proteins). We know now that PRRs can also be recruited and activated upon recognition of endogenous stress signals such as molecules released upon self-cell death (e.g. ATP and HMGB1). Natural endo/exogenous or synthetic PRRs agonists have notably the ability to activate phagocytosis and antigen presentation by myeloid cells residing in the tumor micro-environment. In pre-clinical models, these PRRs agonists have also been shown to overcome the resistance to T-cell targeted immune checkpoints anti-CTLA-4 and anti-PD-1/PD-L1. This manuscript reviews the current knowledge on this major family of immune receptors and the molecules targeting them which are currently in clinical development.

TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16(INK4a) protein expression.

Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16(INK4a) was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16(INK4a) post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis.

Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay.

Monitoring functional competence of immune cell populations in clinical routine represents a major challenge. We developed a whole-blood assay to monitor functional competence of peripheral innate immune cells including NK cells, dendritic and monocyte cell subsets through their ability to produce specific cytokines after short-term stimulation, detected through intra-cytoplasmic staining and multi-parametric flow-cytometry. A PMA/ionomycin T cell activation assay complemented this analysis. Comparing cohorts of healthy women and breast cancer (BC) patients at different stages, we identified significant functional alteration of circulating immune cells during BC progression prior to initiation of treatment. Of upmost importance, as early as the localized primary tumor (PT) stage, we observed functional alterations in several innate immune populations and T cells i.e. (i) reduced TNFα production by BDCA-1+ DC and non-classical monocytes in response to Type-I IFN, (ii) a strong drop in IFNγ production by NK cells in response to either Type-I IFN or TLR7/8 ligand, and (iii) a coordinated impairment of cytokine (IL-2, IFNγ, IL-21) production by T cell subpopulations. Overall, these alterations are further accentuated according to the stage of the disease in first-line metastatic patients. Finally, whereas we did not detect functional modification of DC subsets in response to TLR7/8 ligand, we highlighted increased IL-12p40 production by monocytes specifically at first relapse (FR). Our results reinforce the importance of monitoring both innate and adaptive immunity to better evaluate dysfunctions in cancer patients and suggest that our whole-blood assay will be useful to monitor response to treatment, particularly for immunotherapeutic strategies.

ELYPSE-7: a randomized placebo-controlled phase IIa trial with CYT107 exploring the restoration of CD4+ lymphocyte count in lymphopenic metastatic breast cancer patients.

BACKGROUND: Lymphopenia is a predictive factor for hematological toxicity, progression and early death in advanced cancers including metastatic breast cancer (MBC). CYT107 is a recombinant interleukin 7 (IL-7) (Cytheris, now Revimmune), well tolerated and able to expand lymphocyte pool in humans. The aims of this study were to determine the optimal schedule to deliver CYT107 and to assess its effect on clinical end points. PATIENT AND METHODS: This placebo-controlled, double blind, phase IIa was conducted in MBC patients with <1500/µl lymphocytes treated with capecitabine. Using a 2-by-2 factorial design, 20 patients were randomly allocated to four arms to receive (i) before chemotherapy: CYT107 or placebo; then (ii) during chemotherapy: CYT107 or placebo. The primary end point was CD4+ count changes before and during chemotherapy. Secondary end points were hematological toxicity, safety, overall response, progression-free survival (PFS) and overall survival (OS). Quantification and functional competence of circulating immune cells were also assessed. RESULTS: When administered before chemotherapy, CYT107 induced a significant increase of CD4+ [+148.1% in CYT107 versus +9.9% in placebo groups, (Wilcoxon, P = 0.002)] and CD8+ T-cell counts, including both naïve and memory subsets. When CYT107 was administered during chemotherapy, the magnitude of CD4+ and CD8+ increase was less important. No modulation of immune cell functional competence was observed. CYT107 was well tolerated with no related ≥grade 3 adverse events except 1 fatal suspected unexpected serious adverse reaction (SUSAR) of uncertain relationship. Of the 12 cases evaluable for response, 6 of 7 patients (86%) receiving CYT107 before chemotherapy achieved a response or stabilization, whereas two of five patients (40%) receiving placebo achieved the same result. No significant difference was observed for PFS or OS. CONCLUSION: In lymphopenic MBC, CYT107 increases CD4+ and other T-cell subset counts without altering their function. A larger clinical trial to demonstrate its impact on clinical outcome is warranted. CLINICALTRIALSGOV IDENTIFIER: NCT01362107.

[Spontaneous regression of breast cancer after biopsy. About two cases]. / Régression spontanée de cancers mammaires après biopsie : à propos de deux cas.

We report two cases of spontaneous regression of breast cancer occurred in our institution. It was about a 34- and an 82-year-old patient. The examination revealed a palpable nodule. In both cases, biopsy revealed a negative immunohistochemical staining for estrogen and progesterone receptors, and HER-2 negative. Histological analysis of the surgical specimen found fibrous tissue rearrangement without carcinoma. In the first case, a sarcoidosis was diagnosed at the same time, the mediastinal nodes mimicked a metastatic cancer. These cases illustrate a rare phenomenon. The main hypothesis is a carcinoma- directed immune response triggered by the biopsy.

Autoantibodies to endostatin in patients with breast cancer: correlation to endostatin levels and clinical outcome.

Circulating autoantibodies to self-antigens overexpressed by cancer cells are common in cancer patients. As specific proteins are expressed during neoangiogenesis, a similar phenomenon might occur with particular antigens of tumour vessels. Collagen XVIII, from which endostatin is cleaved, is highly expressed in the perivascular basement membrane of tumour-associated blood vessels and autoantibodies to endostatin have been reported in cancer patients. The present study analyses the incidence of naturally occurring autoantibodies to endostatin in the sera of breast cancer patients and their relation to endostatin serum levels and patient clinical outcome. Serum samples from 36 patients with localised breast cancer and 59 patients with a fully documented history of metastatic breast cancer were used. The immunoreactivity of serum samples was tested against purified recombinant human endostatin and endostatin levels were determined by immunoassay. We could detect anti-endostatin antibodies in the sera of 66% of the patients with localised disease and 42% of the patients with metastatic disease (P=0.03). There was no correlation between the presence of antibodies to endostatin and circulating levels of endostatin. The detection of autoantibodies to endostatin was associated with better prognosis in metastatic breast cancer patients (median survival time: 20 vs 8 months, P = 0.03), as was the presence of low levels of serum endostatin (median survival time: 20 vs 9 months, P = 0.007). These results show that a natural immune reaction against endostatin can occur in breast cancer patients. This could have important therapeutic implications with regard to endostatin therapy and raises the question of a possible role of this humoral reaction against endostatin in the neoplastic process.

Insulin-Like Growth Factor (IGF) family and prostate cancer.

There is abundant in vitro, animal and epidemiologic evidence to suggest that the Insulin-Like Growth Factor (IGF) family is a multi-component network of molecules which is involved in the regulation of both physiological and pathological growth processes in prostate. The IGF family plays a key role in cellular metabolism, differentiation, proliferation, transformation and apoptosis, during normal development and malignant growth. This family also seem essential in prostate cancer bone metastases, angiogenesis and androgen-independent progression. Therapeutic alternatives in men with progressive prostate cancer after androgen ablation are very limited. More effective therapies are needed for these patients. Pharmacologic interventions targeting the IGF family are being devised. Such strategies include reduction of IGF-I levels (growth hormone-releasing hormone antagonists, somatostatin analogs), reduction of functional IGF-I receptor levels (antisense oligonucleotides, small interfering RNA), inhibition of IGF-IR and its signalling (monoclonal antibodies, small-molecule tyrosine kinase inhibitors) and Insulin-Like Growth Factor Binding Proteins.

[Dendritic cells and gliomas: a hope in immunotherapy?]. / Cellules dendritiques et gliomes: un espoir en immunothérapie?

Immunotherapy has been explored for several decades to try to improve the prognosis of gliomas, but until recently no therapeutic benefit has been achieved. The discovery of dendritic cells, the most potent professional antigen presenting cells to initiate specific immune response, and the possibility of producing them ex vivo gave rise to new protocols of active immunotherapy. In oncology, promising experimental and clinical therapeutic results were obtained using these dendritic cells loaded with tumor antigen. Patients bearing gliomas have deficit antigen presentation making this approach rational. In several experimental glioma models, independent research teams have showed specific antitumor responses using these dendritic cells. Phase I/II clinical trials have demonstrated the feasibility and the tolerance of this immunotherapeutic approach. In neuro-oncology, the efficiency of such an approach remains to be established, similarly in oncology where positive phase III studies are missing. Nevertheless, dendritic cells comprise a complex network which is only partially understood and capable of generating either immunotolerance or immune response. Numerous parameters remain to be explored before any definitive conclusion about their utility as an anticancer weapon can be drawn. It seems however logical that immunotherapy with dendritic cells could prevent or delay tumor recurrence in patients with minor active disease. A review on glioma and dendritic cells is presented.

In situ leukemic plasmacytoid dendritic cells pattern of chemokine receptors expression and in vitro migratory response.

Plasmacytoid dendritic cell (PDC) leukemia/lymphoma is a rare neoplasm presenting cutaneous lesions at the time of diagnosis, followed by dissemination to bone marrow, lymph nodes, and other lymphoid and nonlymphoid organs. Since these leukemic counterparts of human PDC are similar to normal PDC, we studied their chemokine receptor equipment and their migratory capacities. We found both in skin lesions and in invaded lymph nodes an expression by tumor cells of CXCR3, CXCR4, and CCR7, and the concomitant expression by cells in the microenvironment of their respective ligands CXCL9, CXCL12, and CCL19. Moreover, flow cytometry phenotype of leukemic PDC (LPDC) revealed an unexpected expression of CCR6. We show that fresh tumor cells are able to migrate in response to CXCR4, CCR2, CCR5, CCR6, and CCR7 ligands, and the ability of CXCR3 ligands to increase the responsiveness to CXCL12. IL-3- or virus-induced activation of LPDC leads to downregulation of CXCR3 and CXCR4, and upregulation of CCR7, associated with the loss of response to CXCL12, and the acquisition of sensitivity to CCL19. Altogether, these results suggest that the preferential accumulation of LPDC in the skin or lymph nodes could be orchestrated by CXCR3, CXCR4, CCR6, and CCR7 ligands, found in nontumoral structures of invaded organs.

Prognostic value of serum levels of interleukin 6 and of serum and plasma levels of vascular endothelial growth factor in hormone-refractory metastatic breast cancer patients.

Prediction of survival for patients with metastatic breast cancer is often inaccurate and may be helped by new biological parameters. Tumour growth being angiogenesis-dependent, it has been hypothesised that the assessment of angiogenic factor production might reflect the clinical behaviour of cancer progression. This study was designed to investigate the clinical significance of vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) in hormone-refractory metastatic breast cancer. Serum and plasma concentrations of VEGF and serum concentration of IL-6 were measured in 87 patients with a fully documented history of metastatic breast cancer using an enzyme-linked immunoassay. All patients had detectable levels of VEGF, whereas 39% patients had detectable serum levels of IL-6. There was a positive correlation between IL-6 levels and the theoretical VEGF load of platelets (P<0.001). The presence of high levels of serum IL-6, but not VEGF, was significantly correlated to a shorter survival. In a multivariate analysis along with clinical prognostic parameters, serum IL-6 was identified as an independent adverse prognostic variable for overall survival (P&<0.001). These results indicate that serum IL-6 levels correlate to poor survival in patients with hormone-refractory metastatic breast cancer. Vascular endothelial growth factor serum and plasma levels are not useful indicators of prognosis for these patients.

Clearance of radiation-induced apoptotic lymphocytes: ex vivo studies and an in vitro co-culture model.

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose-time-effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose-time-effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.

IL-10 induces CCR6 expression during Langerhans cell development while IL-4 and IFN-gamma suppress it.

Immune responses are initiated by dendritic cells (DC) that form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells colonizing epithelial surfaces. We have recently shown that macrophage-inflammatory protein-3alpha/CCL20, a chemokine secreted by epithelial cells, induces the selective migration of LC among DC populations. In this study, we investigated the effects of cytokines on the expression of the CCL20 receptor, CCR6, during differentiation of LC. We found that both IL-4 and IFN-gamma blocked the expression of CCR6 and CCL20 responsiveness at different stages of LC development. The effect of IL-4 was reversible and most likely due to the transient blockade of LC differentiation. In contrast, IFN-gamma-induced CCR6 loss was irreversible and was concomitant to the induction of DC maturation. When other cytokines involved in DC and T cell differentiation were tested, we found that IL-10, unlike IL-4 and IFN-gamma, maintained CCR6 expression. The effect of IL-10 was reversible and upon IL-10 withdrawn, CCR6 was lost concomitantly to final LC differentiation. In addition, IL-10 induced the expression of CCR6 and responsiveness to CCL20 in differentiated monocytes that preserve their ability to differentiate into mature DC. Finally, TGF-beta, which induces LC differentiation, did not alter early CCR6 expression, but triggered its irreversible down-regulation, in parallel to terminal LC differentiation. Taken together, these results suggest that the recruitment of LC at epithelial surface might be suppressed during Th1 and Th2 immune responses, and amplified during regulatory immune responses involving IL-10 and TGF-beta.

Selective attraction of naive and memory B cells by dendritic cells.

In this study, we investigate whether dendritic cells (DC), known to interact directly with T and B cells, might also contribute to the recruitment of B cells through the production of chemotactic factors. We found that B cells responded to several chemokines (CXCL12, CCL19, CCL20, and CCL21), which can be produced by DC upon activation. In addition, supernatant from DC (SNDC) potently and selectively attracted naive and memory B cells but not germinal center (GC) B cells or other lymphocytes (CD4(+), CD8(+) T cells or NK cells). Production of this activity was restricted to DC and was not increased following DC activation by LPS or CD40 ligand. Surprisingly, the B-cell chemotactic response to SNDC was insensitive to pertussis toxin treatment. In addition, the chemotactic factor(s) appeared resistant to protease digestion and highly sensitive to heat. This suggested that the DC chemotactic factor(s) is different from classical chemoattractants and does not involve G(alpha(i)) proteins on the responding B lymphocytes. It is interesting that SNDC was able to synergize with several chemokines to induce massive migration of B lymphocytes. These observations show that DC spontaneously produce factors that, alone or in cooperation with chemokines, specifically regulate B-cell migration, suggesting a key role of DC in the recruitment or localization of B lymphocytes within secondary lymphoid organs.

[Langerin: a new lectin specific for Langerhans cells induces the formation of Birbeck granules]. / Langerin: une nouvelle lectine spécifique des cellules de Langerhans induit la formation de granules de Birbeck.

Generation of monoclonal antibodies restricted to human dendritic cells generated from CD34+ hematopoietic precursors has enabled the identification of Langerin, a Ca(++)-dependent type II lectin. Only expressed by Langerhans cells, Langerin is responsible for Birbeck granule formation by membrane superimposition and zippering. Furthermore, cell-surface Langerin is rapidly internalized into Birbeck granules, and does not colocalize with MHC class II rich compartments. Langerin gene transfected into mouse fibroblasts induces the formation of Birbeck granule-like structures, that would permit a better understanding of the function of Birbeck granules.

New bromoditerpenes from the red alga Sphaerococcus coronopifolius.

The organic extract of the red alga Sphaerococcus coronopifolius, collected along the Atlantic coast of Morocco, was tested for biological activities and exhibited antibiotic and antimalarial activities. Two new bromoditerpenes have been isolated from S. coronopifolius, sphaerolabdadiene-3,14-diol (1) and bromosphaerone (2), along with the known compounds 12S-hydroxybromosphaerodiol (3) and sphaerococcenol A (4). Bromosphaerone and 12S-hydroxybromosphaerodiol showed antibacterial activity against the Gram-positive bacterium species Staphylococcus aureus with a minimum inhibitory concentration of 0.104 and 0.146 microM, respectively. Sphaerococcenol A (4) was responsible for the antimalarial activity of the extract, against the chloroquine resistant Plasmodium falsciparum FCB1 strains with an IC(50) of 1 microM. Their structures have been assigned using 1 and 2 D NMR and HRMS.

Expression of macrophage inflammatory protein-3alpha, stromal cell-derived factor-1, and B-cell-attracting chemokine-1 identifies the tonsil crypt as an attractive site for B cells.

The expression of 3 lymphoid chemokines-macrophage inflammatory protein-3alpha (MIP-3alpha), stromal cell-derived factor-1 (SDF-1), and B-cell-attracting chemokine-1 (BCA-1)-in the tonsil and the possible correlation between their sites of expression and B-cell localization within this tissue were studied. The results show that all 3 chemokines are produced in the crypts but differ by the nature of the cells that produce them and their location within the crypt. SDF-1 and MIP-3alpha are produced by epithelial cells, but their secretion is mutually exclusive. Both MIP-3alpha- and SDF-1-expressing cells are in close contact with memory B cells. By contrast, BCA-1-producing cells in the crypt are not epithelial and form clusters colocalized with plasma cells. Altogether, these data suggest that the chemokines produced in the tonsillar crypt may (1) attract memory B cells to antigen and (2) recruit and retain plasma cells and memory B cells within the supportive epithelial microenvironment of the crypt. (Blood. 2001;97:3992-3994)