Luigi Laghi

Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression‐based stratification. These parameters rely on tumour‐cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM‐I (TNM‐Immune).

Cancer classification using the Immunoscore: a worldwide task force

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the ‘Immunoscore’ into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Reduced likelihood of metastases in patients with microsatellite-unstable colorectal cancer

Purpose: The outcome of patients with colorectal cancer is more favorable when the tumor exhibits high-frequency microsatellite instability (MSI). Although associated with earlier-stage tumors, MSI has been proposed as an independent predictor of survival. We tested the prognostic value of MSI in a large series of patients diagnosed with colorectal cancer in the last decade. Experimental Design: The survival of 893 consecutive patients with colorectal cancer characterized by microsatellite status was analyzed. The 89 (10%) patients with MSI cancer were classified according to tumor mismatch repair (MMR) defect, MMR germ-line mutation, hMLH1 and p16 promoter methylation, BRAF and K-ras mutations, and frameshifts of target genes. Results: The colorectal cancer–specific survival was significantly (P = 0.02) better in patients with MSI cancer than in those with stable tumor (MSS). MSI did not predict a significantly lower risk of cancer-related death if tumor stage was included in the multivariate analysis [hazard ratio, 0.72; 95% confidence interval (95% CI), 0.40-1.29; P = 0.27]. Instead, MSI was strongly associated with a decreased likelihood of lymph node (odds ratio, 0.31; 95% CI, 0.17-0.56; P < 0.001) and distant organ (odds ratio, 0.13; 95% CI, 0.05-0.33; P < 0.001) metastases at diagnosis, independently of tumor pathologic features. Molecular predictors of reduced metastatic risk, and then of more favorable prognosis, included TGFβRII mutation for all MSI tumors, hMSH2 deficiency for hereditary non-polyposis colorectal cancer, and absence of p16 methylation for sporadic hMLH1-deficient cancers. Conclusions: Tumor MSI is a stage-dependent predictor of survival in patients with colorectal cancer. The decreased likelihood of metastases in patients with MSI cancer is associated with specific genetic and epigenetic changes of the primary tumor.

The Chemokine Receptor CX3CR1 Is Involved in the Neural Tropism and Malignant Behavior of Pancreatic Ductal Adenocarcinoma

Tumor perineural dissemination is a hallmark of human pancreatic ductal adenocarcinoma (PDAC) and represents a major source of local tumor recurrence after surgery. In this study, we provide in vitro and in vivo evidence that the chemokine receptor CX3CR1 may be involved in the neurotropism of PDAC cells to local peripheral nerves. Neoplastic cells from PDAC cell lines and surgical specimens express the chemokine receptor CX3CR1, absent in normal pancreatic ducts. Its unique ligand, the transmembrane chemokine CX3CL1, is expressed by neurons and nerve fibers. CX3CR1 + PDAC cell lines migrated in response to human recombinant CX3CL1 and specifically adhered to CX3CL1-expressing cells of neural origin via mechanisms involving activation of G proteins, beta1 integrins, and focal adhesion kinase. In vivo experiments with transplanted PDAC showed that only CX3CR1-transfected tumor cells infiltrated the local peripheral nerves. Immunohistochemistry of CX3CR1 in PDAC specimens revealed that 90% of the samples were positive with a heterogeneous pattern of expression. High receptor score was significantly associated with more prominent tumor perineural infiltration evaluated histologically (P = 0.026). Regression analyses (univariate and multivariate) showed that high CX3CR1 expression and perineural invasion were strongly associated with local and earlier tumor recurrence (P = 0.007). Collectively, this study shows that the CX3CR1 receptor may be involved in PDAC tumor neurotropism and is a relevant and independent risk factor to predict an early local tumor relapse in resected patients. Thus, the CX3CR1-CX3CL1 axis could represent a valuable therapeutic target to prevent tumor perineural dissemination in pancreatic cancer.

PTX3 Is an Extrinsic Oncosuppressor Regulating Complement-Dependent Inflammation in Cancer

PTX3 is an essential component of the humoral arm of innate immunity, playing a nonredundant role in resistance against selected microbes and in the regulation of inflammation. PTX3 activates and regulates the Complement cascade by interacting with C1q and with Factor H. PTX3 deficiency was associated with increased susceptibility to mesenchymal and epithelial carcinogenesis. Increased susceptibility of Ptx3(-/-) mice was associated with enhanced macrophage infiltration, cytokine production, angiogenesis, and Trp53 mutations. Correlative evidence, gene-targeted mice, and pharmacological blocking experiments indicated that PTX3 deficiency resulted in amplification of Complement activation, CCL2 production, and tumor-promoting macrophage recruitment. PTX3 expression was epigenetically regulated in selected human tumors (e.g., leiomyosarcomas and colorectal cancer) by methylation of the promoter region and of a putative enhancer. Thus, PTX3, an effector molecule belonging to the humoral arm of innate immunity, acts as an extrinsic oncosuppressor gene in mouse and man by regulating Complement-dependent, macrophage-sustained, tumor-promoting inflammation.

Mad-1 Is the Exclusive JC Virus Strain Present in the Human Colon, and Its Transcriptional Control Region Has a Deleted 98-Base-Pair Sequence in Colon Cancer Tissues

ABSTRACT JC virus (JCV), along with other members of the polyomavirus family, encodes a class of highly conserved proteins, T antigens, that are capable of inducing aneuploidy in cultured cells. We have previously isolated T-antigen DNA variants of JCV from both colon cancer tissues and the corresponding nonneoplastic gastrointestinal tissues, raising new questions about the role of JCV in the development of chromosomal instability of the colon. Based on the sequence of the transcriptional control region (TCR), JCV can be classified as archetype or tandem repeat variants. Among the latter, Mad-1, the prototype virus first isolated from a patient with progressive multifocal leukoencephalopathy, is characterized by lacking the 23- and 66-bp sequences that are present in the archetype and by duplication of a 98-bp sequence. In this study, we evaluated differences in the TCR of JCV isolated from colon cancer tissues and nonneoplastic epithelium. To characterize JCV variants, we first treated eight pairs of DNA samples from colon cancers and noncancerous tissue with topoisomerase I and then amplified and cloned the JCV TCR. We obtained 285 recombinant clones from the JCV TCR, 157 from nonneoplastic samples, and 128 from colon cancer tissues. Of these clones, 262 spanned the length of the JCV Mad-1 TCR: 99.3% from nonneoplastic samples and 82.8% from colon cancer tissues. In sequencing 54 clones in both directions, we did not find archetype JCV either in the nonneoplastic tissue or in the cancer samples. From all colon cancer tissues, 18 clones had a deletion of one 98-bp tandem repeat. This deleted strain was not detected in any of the nonneoplastic tissues (14 versus 0% [χ2 = 23.6;P < 0.001]). Our study demonstrates that the only JCV strain present in the human colon is Mad-1, and the variant with a single 98-bp sequence is found exclusively in the cancer tissues. This strain may be involved in the development of chromosomal instability.

Differences and evolution of the methods for the assessment of microsatellite instability

Microsatellite instability (MSI) originates from the systematic accumulation of uncorrected deletion/insertion in repetitive DNA tracts in cancer cells with a deficient mismatch repair system. Among colorectal cancers, the MSI signature identifies hereditary cases arising in patients with germline mutations in hMLH1, hMSH2, PMS2 and a fraction of those with hMSH6 mutations, as well as sporadic cancers with epigenetic hMLH1 promoter hypermethylation. Considering the specific pathogenesis, pathological features, natural history and response to 5-fluoro-uracil-based chemotherapy of the MSI cancers, confusion about the genetic markers for MSI recognition seems surprising. In this clinically relevant field, an agreement has not been reached concerning the use of di- or mononucleotide markers for MSI assessment. The Revised Bethesda Guidelines still recommend a panel of markers consisting of mono- and dinucleotides, despite being questioned whether it is congruous to continue to use dinucleotide markers for MSI identification. In any event, no single marker is accurate enough for MSI testing, and an awareness of their pros and cons is required for proper interpretation of results. In recent years, several papers have reported different prevalence of MSI in unrelated series, largely depending on the detection and classification method, suggesting that MSI test interpretation also requires the understanding of the phenomenon rather than simply the crude satisfaction of panel recommendations. Inaccuracies can otherwise lead to under- or overdiagnosis and inaccurate disease classification, which always have a negative impact on the clinical practice of medicine.

The tumor microenvironment of colorectal cancer: stromal TLR-4 expression as a potential prognostic marker

BackgroundColorectal cancer can be efficiently treated when found at early stages, thus the search for novel markers is of paramount importance. Since inflammation is associated with cancer progression and angiogenesis, we investigated expression of cytokines like IL-6 and other mediators that play a key role in the innate immune system, in particular toll like receptor 4 (TLR4), in the microenvironment of lesions from different stages of colon disease progression, from ulcerative colitis to adenoma and adenocarcinoma to find useful markers.MethodsThe presence of inflammatory cells and expression of key cytokines involved in the inflammation process were quantified by immunohistochemistry in specific tissue compartments (epithelial, stromal, endothelial) by immunohistochemistry. A murine azoxymethane/dextran sulfate model in which Tir8, a negative regulator of the inflammatory response, was ablated was used to confirm the clinical observations. 116 Archival tissue samples from patients with different stages of colorectal disease: 13 cases of ulcerative colitis (UC), 34 tubular or tubulo-villous adenomas (AD), and 53 infiltrating adenocarcinomas. 16 specimens of healthy mucosa surgically removed with the cancerous tissue were used as a control.ResultsThe differences between healthy tissues and the diverse lesions was characterized by a marked inflammatory-angiogenic reaction, with significantly (P < 0.05) higher numbers of CD68, CD15, and CD31 expressing cells in all diseased tissues that correlated with increasing grade of malignancy. We noted down-regulation of a potential modulator molecule, Hepatocyte Growth Factor, in all diseased tissues (P < 0.05). TLR-4 and IL6 expression in the tumor microenvironment were associated with adenocarcinoma in human samples and in the murine model. We found that adenocarcinoma patients (pT1-4) with higher TLR-4 expression in stromal compartment had a significantly increased risk in disease progression. In those patients with a diagnosis of pT3 (33 cases) colon cancer, those with very high levels of TLR-4 in the tumor stroma relapsed significantly earlier than those with lower expression levels.ConclusionsThese data suggest that high TLR-4 expression in the tumor microenvironment represents a possible marker of disease progression in colon cancer.

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.

Genetic and epigenetic changes in primary metastatic and nonmetastatic colorectal cancer

Colorectal cancer (CRC) develops as multistep process, which involves genetic and epigenetic alterations. K-Ras, p53 and B-Raf mutations and RASSF1A, E-Cadherin and p16INK4A promoter methylation were investigated in 202 CRCs with and without lymph node and/or liver metastasis, to assess whether gene abnormalities are related to a metastogenic phenotype. K-Ras, B-Raf and p53 mutations were detected in 27, 3 and 32% of the cases, with K-Ras mutations significantly associated with metastatic tumour (P=0.019). RASSF1A, E-Cadherin and p16INK4A methylation was documented in 20, 44 and 33% of the cases with p16INK4A significantly associated with metastatic tumours (P=0.001). Overall, out of 202 tumours, 34 (17%) did not show any molecular change, 125 (62%) had one or two and 43 (21%) three or more. Primary but yet metastatic CRCs were prevalent in the latter group (P=0.023) where the most frequent combination was one genetic (K-Ras in particular) and two epigenetic alterations. In conclusion, this analysis provided to detect some molecular differences between primary metastatic and nonmetastatic CRCs, with K-Ras and p16INK4A statistically altered in metastatic tumours; particular gene combinations, such as coincidental K-Ras mutation with two methylated genes are associated to a metastogenic phenotype.