Chantal Mengus

Tumor infiltration by FcγRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan–Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearmans rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6–0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7–0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3–0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64− HLA‐DR‐myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.

“In vitro” 3D models of tumor-immune system interaction ☆

Interaction between cancer cells and immune system critically affects development, progression and treatment of human malignancies. Experimental animal models and conventional in vitro studies have provided a wealth of information on this interaction, currently used to develop immune-mediated therapies. Studies utilizing three-dimensional culture technologies have emphasized that tumor architecture dramatically influences cancer cell-immune system interaction by steering cytokine production and regulating differentiation patterns of myeloid cells, and decreasing the sensitivity of tumor cells to lymphocyte effector functions. Hypoxia and increased production of lactic acid by tumor cells cultured in 3D architectures appear to be mechanistically involved. 3D culture systems could be further developed to (i) include additional cell partners potentially influencing cancer cell-immune system interaction, (ii) enable improved control of hypoxia, and (iii) allow the use of freshly derived clinical cancer specimens. Such advanced models will represent new tools for cancer immunobiology studies and for pre-clinical assessment of innovative treatments.

Elevated levels of circulating IL-7 and IL-15 in patients with early stage prostate cancer

BackgroundChronic inflammation has been suggested to favour prostate cancer (PCA) development. Interleukins (IL) represent essential inflammation mediators. IL-2, IL-7, IL-15 and IL-21, sharing a common receptor γ chain (c-γ), control T lymphocyte homeostasis and proliferation and play major roles in regulating cancer-immune system interactions. We evaluated local IL-2, IL-7, IL-15 and IL-21 gene expression in prostate tissues from patients with early stage PCA or benign prostatic hyperplasia (BPH). As control, we used IL-6 gene, encoding an IL involved in PCA progression. IL-6, IL-7 and IL-15 titres were also measured in patients sera.MethodsEighty patients with BPH and 79 with early (1 to 2c) stage PCA were enrolled. Gene expression in prostate tissues was analyzed by quantitative real-time PCR (qRT-PCR). Serum IL concentrations and acute phase protein titres were evaluated by ELISA. Mann-Whitney, Wilcoxon and χ2 tests were used to compare IL gene expression and serum titers in the two groups of patients. Receiver operating characteristic (ROC) curves were constructed to evaluate the possibility to distinguish sera from different groups of patients based on IL titers.ResultsIL-2 and IL-21 gene expression was comparably detectable, with low frequency and at low extents, in PCA and BPH tissues. In contrast, IL-6, IL-7 and IL-15 genes were expressed more frequently (p < 0.0001, p = 0.0047 and p = 0.0085, respectively) and to significantly higher extents (p = 0.0051, p = 0.0310 and p = 0.0205, respectively) in early stage PCA than in BPH tissues. Corresponding proteins could be detected to significantly higher amounts in sera from patients with localized PCA, than in those from patients with BPH (p = 0.0153, p = 0.0174 and p = 0.0064, respectively). Analysis of ROC curves indicates that IL-7 (p = 0.0039), but not IL-6 (p = 0.2938) or IL-15 (p = 0.1804) titres were able to distinguish sera from patients with malignancy from those from patients with benign disease. Serum titres of C reactive (CRP), high mobility group B1 (HMGB1) and serum amyloid A (SAA) acute phase proteins were similar in both groups of patients.ConclusionsExpression IL-7 and IL-15 genes in prostate tissues and corresponding serum titres are significantly increased in patients with early stage PCA as compared with patients with BPH.

Klf4 transcription factor is expressed in the cytoplasm of prostate cancer cells.

BACKGROUNDnCancer initiation and progression might be driven by small populations of cells endowed with stem cell-like properties. Here we comparatively addressed the expression of genes encoding putative stemness regulators including c-Myc, Klf4, Nanog, Oct4A and Sox2 genes in benign prostatic hyperplasia (BPH) and prostate cancer (PCA).nnnMETHODSnFifty-eight PCA and thirty-nine BPH tissues samples were used for gene expression analysis, as evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of specific Klf4 isoforms was tested by conventional PCR. Klf4 specific antibodies were used for protein detection in a tissue microarray including 404 prostate samples.nnnRESULTSnNanog, Oct4A and Sox2 genes were comparably expressed in BPH and PCA samples, whereas c-Myc and Klf4 genes were expressed to significantly higher extents in PCA than in BPH specimens. Immunohistochemical studies revealed that Klf4 protein is detectable in a large majority of epithelial prostatic cells, irrespective of malignant transformation. However, in PCA, a predominantly cytoplasmic location was observed, consistent with the expression of a differentially spliced Klf4α isoform.nnnCONCLUSIONnKlf4 is highly expressed at gene and protein level in BPH and PCA tissues but a cytoplasmic location of the specific gene product is predominantly detectable in malignant cells. Klf4 location might be of critical relevance to steer its functions during oncogenesis.

Bioreactor-engineered cancer tissue-like structures mimic phenotypes, gene expression profiles and drug resistance patterns observed “in vivo”

Anticancer compound screening on 2D cell cultures poorly predicts in vivo performance, while conventional 3D culture systems are usually characterized by limited cell proliferation, failing to produce tissue-like-structures (TLS) suitable for drug testing. We addressed engineering of TLS by culturing cancer cells in porous scaffolds under perfusion flow. Colorectal cancer (CRC) HT-29 cells were cultured in 2D, on collagen sponges in static conditions or in perfused bioreactors, or injected subcutaneously in immunodeficient mice. Perfused 3D (p3D) cultures resulted in significantly higher (p < 0.0001) cell proliferation than static 3D (s3D) cultures and yielded more homogeneous TLS, with morphology and phenotypes similar to xenografts. Transcriptome analysis revealed a high correlation between xenografts and p3D cultures, particularly for gene clusters regulating apoptotic processes and response to hypoxia. Treatment with 5-Fluorouracil (5-FU), a frequently used but often clinically ineffective chemotherapy drug, induced apoptosis, down-regulation of anti-apoptotic genes (BCL-2, TRAF1, and c-FLIP) and decreased cell numbers in 2D, but only nucleolar stress in p3D and xenografts. Conversely, BCL-2 inhibitor ABT-199 induced cytotoxic effects in p3D but not in 2D cultures. Our findings advocate the importance of perfusion flow in 3D cultures of tumor cells to efficiently mimic functional features observed in vivo and to test anticancer compounds.

MAGE‐A10 cancer/testis antigen is highly expressed in high‐grade non‐muscle‐invasive bladder carcinomas

Bladder cancer is a common urinary malignancy and a prevalent cause of cancer‐related death. Current therapies of early stage non‐muscle‐invasive bladder cancer (NMIBC) are frequently associated with undesirable toxicities and recurrence. Active antigen‐specific immunotherapy may provide a valid therapeutic option for patients with NMIBC. Cancer‐testis antigens (CTA) expressed in various tumour types and in a limited range of healthy tissues may represent potential targets for specific immunotherapy. MAGE‐A10 is probably the most immunogenic antigen of the MAGE‐A family. We evaluated the expression of MAGE‐A10 in NMIBC. Seventy‐nine patients undergoing surgical treatment for NMIBC were enrolled in the study. MAGE‐A10 gene expression was assessed by quantitative real‐time polymerase chain reaction. Immunohistochemistry was performed on paraffin‐embedded sections. MAGE‐A10 gene was specifically expressed in one‐third of NMIBC (n = 24: 32.43%). Gene expression was correlated with high tumour grade. MAGE‐A10 protein was exclusively detectable in nuclei of tumour cells. More importantly, MAGE‐A10 protein was also more frequently detectable in high‐grade tumours (p = 0.0001) and in stage T1 tumours invading subepithelial tissue or lamina propria (p = 0.01). A strong correlation between MAGE‐A10 staining score and tumour grade and stage could accordingly be observed. These data indicate that MAGE‐A10 expression is a feature of aggressive NMIBC and might be used as a novel target for specific immunotherapy of these cancers.

Differential Patterns of Large Tumor Antigen-Specific Immune Responsiveness in Patients with BK Polyomavirus-Positive Prostate Cancer or Benign Prostatic Hyperplasia

ABSTRACT The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor β1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4+ CD25+(high) CD127− FoxP3+ T cell population with an effector memory phenotype (CD103+) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4+ CD25− T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV+ PCa.

Immunohistochemical analysis of the expression of MAGE-A and NY-ESO-1 cancer/testis antigens in diffuse large B-cell testicular lymphoma

BackgroundPrimary testicular lymphoma (PTL) is a rare and lethal disease. The most common histological subtype is diffuse large B-cell lymphoma (DLBCL). Standard treatments are frequently ineffective. Thus, the development of novel forms of therapy is urgently required. Specific immunotherapy generating immune responses directed against antigen predominantly expressed by cancer cells such as cancer-testis antigens (CTA) may provide a valid alternative treatment for patients bearing PTL, alone or in combination with current therapies.MethodsThree monoclonal antibodies (mAbs), 77B recognizing MAGE-A1, 57B recognizing an epitope shared by multiple MAGE-A CTA (multi-MAGE-A specific) and D8.38 recognizing NY-ESO-1/LAGE-1 were used for immunohistochemical staining of 27 PTL, including 24 DLBCL.ResultsExpression of MAGE-A1 was infrequently detectable in DLBCL specimens (12.50%), whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained the cytoplasms of tumor cells in DLBCL specimens with higher frequencies (54.17% and 37.50%, respectively) with different expression levels.ConclusionsThese results suggest that MAGE-A and NY-ESO-1/LAGE-1, possibly in combination with other CTA, might be used as targets for specific immunotherapy in DLBCL.

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class‐I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class‐I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV‐US12) or together with endoplasmic reticulum (ER)‐targeted Melan‐A/MART‐127–35 model tumor epitope (rVV‐MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV‐US12, display a decreased ability to present TAP dependent MHC class‐I restricted viral antigens to CD8+ T‐cells. While HLA class‐I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class‐II are unaffected. Characterization of rVV‐MUS12 infected cells demonstrates that over‐expression of a TAP‐independent peptide, partially compensates for ICP47 induced surface MHC class‐I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus‐specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV‐MUS12, as compared to those stimulated by rVV‐MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector‐specific responsiveness.

CD40 ligand-expressing recombinant vaccinia virus promotes the generation of CD8+ central memory T cells

Central memory CD8+ T cells (TCM) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8+ TCM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8+ TCM‐cell generation is challenging, and usually requires CD4+ CD40L+ T‐cell “help” during the priming of naïve CD8+ T cells. We have generated a replication incompetent CD40 ligand‐expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8+ T cells into TCM specific for viral and tumor‐associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild‐type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4+ T cells, a single “in vitro” stimulation of naïve CD8+ T cells by rVV40L‐infected nonprofessional CD14+ antigen presenting cells promotes the rapid generation of viral or tumor associated antigen‐specific CD8+ T cells displaying TCM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8+ mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.