Giuseppe Di Caro

Occurrence of Tertiary Lymphoid Tissue Is Associated with T-Cell Infiltration and Predicts Better Prognosis in Early-Stage Colorectal Cancers

Purpose: Tumor-infiltrating T lymphocytes (TIL) play a key role in the clinical outcome of human colorectal cancer; however, the dynamics of their recruitment along colorectal cancer clinical progression have not been fully elucidated. Tertiary lymphoid tissue (TLT) is an ectopic organized lymph node–like structure that typically forms at sites of chronic inflammation and is involved in adaptive immune responses. Its occurrence in cancer is sporadically documented and its role and clinical relevance is largely unknown. Experimental Design: The occurrence of TLT, the correlation with TILs, and the clinical relevance were evaluated retrospectively, in a cohort study involving a consecutive series of 351 patients with stage II and III colorectal cancer. The role of TLT in lymphocyte recruitment was assessed in a preclinical model of colorectal cancer. Results: In both human colorectal cancer and in a murine model of colorectal cancer, we identified organized TLT, highly vascularized (including high endothelial venules), and correlated with the density of CD3+ TILs. Intravenous injection in mice of GFP splenocytes resulted in homing of lymphocytes to TLT, suggesting an active role of TLT in the recruitment of lymphocytes to tumor areas. Accordingly, TLT density and TIL infiltration correlated and were coordinated in predicting better patients outcome among patients with stage II colorectal cancer. Conclusions: We provide evidence that TLT is associated with lymphocyte infiltration in colorectal cancer, providing a pathway of recruitment for TILs. TLT cooperates with TILs in a coordinated antitumor immune response, when identifying patients with low-risk early-stage colorectal cancer, thus, representing a novel prognostic biomarker for colorectal cancer. Clin Cancer Res; 20(8); 2147–58. ©2014 AACR.

Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy

Objective Tumour-associated macrophages (TAMs) play key roles in tumour progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. Design In a large retrospective cohort study involving 110 patients with pancreatic ductal adenocarcinoma (PDAC), we assessed the density of CD68-TAM immune reactive area (%IRA) at the tumour–stroma interface and addressed their prognostic relevance in relation to postsurgical adjuvant chemotherapy (CTX). In vitro, we dissected the synergism of CTX and TAMs. Results In human PDAC, TAMs predominantly exhibited an immunoregulatory profile, characterised by expression of scavenger receptors (CD206, CD163) and production of interleukin 10 (IL-10). Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumour prognostic significance. High density of TAMs at the tumour–stroma interface positively dictated prognostic responsiveness to CTX independently of T-cell density. Accordingly, in vitro, gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression programme, inhibiting their protumoural effect and switching to an antitumour phenotype. In patients with human PDAC, neoadjuvant CTX was associated to a decreased density of CD206+ and IL-10+ TAMs at the tumour–stroma interface. Conclusions Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX overall directly re-educates TAMs to restrain tumour progression. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide the basis for novel strategies aimed at re-educating macrophages in the context of CTX.

Immune cells: plastic players along colorectal cancer progression

Inflammatory cells are involved in tumour initiation and progression. In parallel, the adaptive immune response plays a key role in fighting tumour growth and dissemination. The double‐edged role of the immune system in solid tumours is well represented in colorectal cancer (CRC). The development and progression of CRC are affected by the interactions between the tumour and the hosts response, occurring in a milieu named tumour microenvironment. The role of immune cells in human CRC is being unravelled and there is a strong interest in understanding their dynamics as to tumour promotion, immunosurveillance and immunoevasion. A better definition of immune infiltration would be important not only with respect to the ‘natural history’ of CRC, but in a clinically relevant perspective in the 21st century, with respect to its post‐surgical management, including chemotherapy responsiveness. While it is becoming established that the amount of tumour‐infiltrating lymphocytes influences the post‐surgical progression of early‐stage CRC, the relevance of this immune parameter as to chemotherapy responsiveness remains to be clarified. Despite recent experimental work supporting the notion that infiltrating immune cells may influence chemotherapy‐mediated tumour cell death, tumour‐infiltrating cells are not employed to identify patients who are more likely to benefit from adjuvant treatment. This review focuses on studies addressing the role of innate and adaptive immune cells along the occurrence and the progression of potentially curable CRC.

Irrelevance of Microsatellite Instability in the Epidemiology of Sporadic Pancreatic Ductal Adenocarcinoma

Background and Aims Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS. Methods MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers. Results Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one. Conclusions MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.

Occurrence and significance of tumor-associated neutrophils in patients with colorectal cancer.

Inflammatory cells are an essential component of the tumor microenvironment. Neutrophils have emerged as important players in the orchestration and effector phase of innate and adaptive immunity. The significance of tumor‐associated neutrophils (TAN) in colorectal cancer (CRC) has been the subject of conflicting reports and the present study was designed to set up a reliable methodology to assess TAN infiltration in CRC and to evaluate their clinical significance. CD66b and myeloperoxidase (MPO) were assessed as candidate neutrophil markers in CRC using immunohistochemistry. CD66b was found to be a reliable marker to identify TAN in CRC tissues, whereas MPO also identified a subset of CD68+ macrophages. CRC patients (n = 271) (Stages I–IV) were investigated retrospectively by computer‐assisted imaging on whole tumor sections. TAN density dramatically decreases in Stage IV patients as compared to Stage I–III. At Cox analysis, higher TAN density was associated with better prognosis. Importantly, multivariate analysis showed that prognostic significance of TAN can be influenced by clinical stage and 5‐fluorouracil(5‐FU)‐based chemotherapy. On separate analysis of Stage III patients (n = 178), TAN density had a dual clinical significance depending on the use of 5‐FU‐based chemotherapy. Unexpectedly, higher TAN density was associated with better response to 5‐FU‐based chemotherapy. Thus, TAN are an important component of the immune cell infiltrate in CRC and assessment of TAN infiltration may help identify patients likely to benefit from 5‐FU‐based chemotherapy. These results call for a reassessment of the role of neutrophils in cancer using rigorous quantitative methodology.

Presence of Twist1-Positive Neoplastic Cells in the Stroma of Chromosome-Unstable Colorectal Tumors

BACKGROUND & AIMS Cancer cells undergo an epithelial-to-mesenchymal transition (EMT) to become invasive, allowing tumors to progress. However, there is no direct evidence that human cancer cells undergo an EMT. In mouse cancer cells, up-regulation of transcription factor Twist1 was shown to promote an EMT. We searched the stroma of human colorectal tumor samples for TWIST1-positive cells with a mesenchymal phenotype and neoplastic genotype. METHODS We measured the expression of TWIST1 in human colorectal cancer (CRC) cell lines and examined the effects of overexpression or knockdown in vitro and in mice. We used immunohistochemistry to measure levels of TWIST1 in 201 colorectal tumor samples. In 20 samples, immunostaining was combined with fluorescence in situ hybridization analyses. Levels of TWIST1 messenger RNA (mRNA) were measured in blood samples from 15 patients. RESULTS TWIST1 was required to maintain the mesenchymal phenotype and invasiveness of the microsatellite-stable CoLo741 cells (which express endogenous TWIST1) and SW480 (expressing transgenic TWIST1). TWIST1 mRNA was not translated in CRC cells with microsatellite instability (HCT116). Syngenic TWIST1-positive colon carcinoma cells (CT26) that invaded tissues surrounding tumors acquired a mesenchymal phenotype. The presence of TWIST1-positive cells in the stroma of human colorectal tumors correlated with microsatellite stability (P = .05), stage IV cancer (P = .02), and disease-free survival time (P < .01). Trisomies of chromosome 7 and/or chromosome 20 were detected in 17 of 20 colorectal tumor samples, each of which contained TWIST1-positive cells with matching chromosomal gains in the tumor stroma (86 of 776 counted cells; 11.1%). No trisomy was observed in TWIST1-negative stromal cells (0 of 1249 cells; P < .001). Levels of TWIST1 mRNA were significantly higher in blood samples from patients with CRC than controls. CONCLUSIONS The stroma of human colorectal tumors contains TWIST1-positive cancer cells with mesenchymal phenotypes. Patients with CRC have higher levels of TWIST1 mRNA than healthy individuals.

Spatial distribution of B cells predicts prognosis in human pancreatic adenocarcinoma

ABSTRACT B-cell responses are emerging as critical regulators of cancer progression. In this study, we investigated the role of B lymphocytes in the microenvironment of human pancreatic ductal adenocarcinoma (PDAC), in a retrospective consecutive series of 104 PDAC patients and in PDAC preclinical models. Immunohistochemical analysis revealed that B cells occupy two histologically distinct compartments in human PDAC, either scatteringly infiltrating (CD20-TILs), or organized in tertiary lymphoid tissue (CD20-TLT). Only when retained within TLT, high density of B cells predicted longer survival (median survival 16.9 mo CD20-TLThi vs. 10.7 mo CD20-TLTlo; p = 0.0085). Presence of B cells within TLT associated to a germinal center (GC) immune signature, correlated with CD8-TIL infiltration, and empowered their favorable prognostic value. Immunotherapeutic vaccination of spontaneously developing PDAC (KrasG12D-Pdx1-Cre) mice with α-enolase (ENO1) induced formation of TLT with active GCs and correlated with increased recruitment of T lymphocytes, suggesting induction of TLT as a strategy to favor mobilization of immune cells in PDAC. In contrast, in an implanted tumor model devoid of TLT, depletion of B cells with an anti-CD20 antibody reinstated an antitumor immune response. Our results highlight B cells as an essential element of the microenvironment of PDAC and identify their spatial organization as a key regulator of their antitumor function. A mindfully evaluation of B cells in human PDAC could represent a powerful prognostic tool to identify patients with distinct clinical behaviors and responses to immunotherapeutic strategies.

Inflammation and prostate cancer: friends or foe?

IntroductionProstate cancer is the most common non-cutaneous malignancy diagnosed in men. Moving from histological observations since a long time, it has been recognized that innate and adaptive immunity actively participates in the pathogenesis, surveillance, and progression of prostate cancer.Materials and methodsA PubMed and Web of Science databases search was performed for studies providing evidence on the roles of the innate and adaptive immunity during the development and progression of prostate cancer.ConclusionsThere are growing evidences that chronic inflammation is involved in the regulation of cellular events in prostate carcinogenesis, including disruption of the immune response and regulation of the tumor microenvironment. This review discusses the role played by the innate and adaptive immune system in the local progression of prostate cancer, and the prognostic information that we can currently understand and exploit.

Mast cells and the liver aging process

It has now ascertained that the clinical manifestations of liver disease in the elderly population reflect both the cumulative effects of longevity on the liver and the generalized senescence of the organism ability to adjust to metabolic, infectious, and immunologic insults. Although liver tests are not significantly affected by age, the presentation of liver diseases such as viral hepatitis may be subtler in the elderly population than that of younger patients.Human immunosenescence is a situation in which the immune system, particularly T lymphocyte function, deteriorates with age, while innate immunity is negligibly affected and in some cases almost up-regulated.We here briefly review the relationships between the liver aging process and mast cells, the key effectors in a more complex range of innate immune responses than originally though.

MSH3 Protein Expression and Nodal Status in MLH1-Deficient Colorectal Cancers

Purpose: Patients with colorectal cancers (CRC) and high microsatellite instability (MSI) have a better outcome than their chromosome-unstable counterpart. Given the heterogeneity of microsatellite-unstable CRCs, we wanted to see whether any MSI-associated molecular features are specifically associated with prognosis. Experimental Design: One hundred and nine MSI-high CRCs were typed for primary mismatch repair (MMR) defect and for secondary loss of MMR proteins. Frameshifts at seven target genes, mutations in the RAS pathway, and methylation at MLH1/CDKN2A promoters were also searched. The interplay of molecular findings with clinicopathologic features and patient survival was analyzed. Results: Of 84 MLH1-deficient CRCs, 31 (36.9%) had MSH3 and 11 (13.1%) had MSH6 loss (P < 0.001), biallelic frameshift mutations at mononucleotide repeats accounting for most (78%) MSH3 losses. As compared with MSH3-retaining cancers, MLH1-deficient tumors with MSH3 loss showed a higher number of mutated target genes (3.94 ± 1.56 vs. 2.79 ± 1.75; P = 0.001), absence of nodal involvement at pathology [N0; OR, 0.11; 95% confidence interval (CI), 0.04–0.43, P < 0.001], and better disease-free survival (P = 0.06). No prognostic value was observed for KRAS status and for MLH1/CDKN2A promoter methylation. The association between MSH3 loss and N0 was confirmed in an independent cohort of 71 MLH1-deficient CRCs (OR, 0.23; 95% CI, 0.06–0.83, P = 0.02). Conclusions: MLH1-deficient CRCs not expressing MSH3 have a more severe MSI, a lower rate of nodal involvement, and a better postsurgical outcome. Clin Cancer Res; 18(11); 3142–53. ©2012 AACR.