Fabiana Tatangelo

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).

Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients

Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade.

Overexpression of Both CXC Chemokine Receptor 4 and Vascular Endothelial Growth Factor Proteins Predicts Early Distant Relapse in Stage II-III Colorectal Cancer Patients

Purpose: CXC chemokine receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF) are implicated in the metastatic process of malignant tumors. However, no data are currently available on the biological relationship between these molecules in colorectal cancer. We studied whether CXCR4 and VEGF expression could predict relapse and evaluated in vitro the contribution of CXCR4 in promoting clonogenic growth, VEGF secretion, and intercellular adhesion molecule-1 (ICAM-1) expression of colorectal cancer cells. Experimental Design: CXCR4 and VEGF were studied in colorectal cancer tissues and in Lovo, HT29, and SW620 colorectal cancer cell lines by immunohistochemistry. Correlations with baseline characteristics of patients and tumors were analyzed by χ2 test. VEGF secretion induced by CXCL12 was measured by ELISA. The effect of CXCL12 on ICAM-1 expression was evaluated by flow cytometry. Clonogenic growth induced by CXCL12 was determined by clonogenic assays. Functional effects induced by CXCL12 were prevented by the administration in vitro of AMD3100, a bicyclam noncompetitive antagonist of CXCR4. Results: Seventy-two patients, seen between January 2003 and January 2004, were studied. CXCR4 was absent in 16 tumors (22.2%); it was expressed in ≤50% of cells in 25 (34.7%) tumors and in >50% of cells in 31 (43.0%) tumors. VEGF was absent in 17 (23.6%) tumors; it was expressed in ≤50% of cells in 16 (22.2%) tumors and in >50% of cells in 39 (54.2%) tumors. There was a significant association between CXCR4 expression and lymph nodal status (P = 0.0393). There were significant associations between VEGF and tumor invasion (P = 0.0386) and lymph nodal involvement (P = 0.0044). American Joint Committee on Cancer stage (P = 0.0016), VEGF expression (P = 0.0450), CXCR4 expression (P = 0.0428), and VEGF/CXCR4 expression (P = 0.0004) had a significant prognostic value for disease-free survival with univariate analysis. The predictive ability of the American Joint Committee on Cancer stage and of the concomitant and high expression of VEGF and CXCR4 was confirmed by multivariate analysis. Prognosis is particularly unfavorable for patients whose primary tumors express CXCR4 and VEGF in >50% of cells (median disease-free survival in relapsed patients, 5.8 months; hazard ratio of relapse, 8.23; 95% confidence interval, 7.24-14.29). In clonogenic assays, CXCL12 (20 ng/mL/d) significantly increased the number of clones in SW620, HT29, and Lovo cells at 7 and 14 days. Again, CXCL12 was able to stimulate VEGF secretion in SW620, HT29, and Lovo cells as well as up-regulated ICAM-1. These effects were prevented by the administration of AMD3100 (1 μmol/L). Conclusions: We have shown that concomitant and high expression of CXCR4 and VEGF is a strong and independent predictor of early distant relapse in colorectal cancer. CXCR4 triggers a plethora of phenomena, including stimulation of clonogenic growth, induction of VEGF release, and ICAM-1 up-regulation. These data support the inhibition of CXCR4 to prevent the development of colorectal cancer metastasis.

Expression of COX-2, mPGE-synthase1, MDR-1 (P-gp), and Bcl-xL: a molecular pathway of H pylori-related gastric carcinogenesis.

Helicobacter pylori up‐regulates cyclo‐oxygenase‐2 (COX‐2) expression, which in turn is involved in tumourigenesis. Recently, a causal link between COX‐2 and multidrug resistance 1 (MDR‐1) gene expression, implicated in cancer chemoresistance, has been demonstrated. Thus, the expression of COX‐2 and the downstream enzyme involved in PGE2 biosynthesis, microsomal PGE‐synthase1 (mPGES1), was correlated with P‐gp, the product of MDR‐1, and the anti‐apoptotic protein, Bcl‐xL, in gastric biopsies from patients with H pylori infection and in patients with gastric cancer. In a retrospective analysis of endoscopic and pathology files, 40 H pylori‐negative patients (Hp−), 50 H pylori‐positive patients who responded to eradication therapy (Hp+R), 84 H pylori‐positive patients who did not respond to eradication therapy (Hp+NR), and 30 patients with gastric cancer (18 intestinal and 12 diffuse types) were selected. COX‐2, mPGES1, P‐gp, and Bcl‐xL were detected by immunohistochemistry. COX‐2, mPGES1, P‐gp, and Bcl‐xL expression was undetectable in gastric mucosa from Hp− patients. By contrast, COX‐2 and mPGES1 expression was detected in 42% and 44% of Hp+R patients, respectively, and in up to 66% (range 63–66%) of Hp+NR patients (p < 0.05). The expression of COX‐2 and mPGES1 correlated significantly (p < 0.0001) with that of P‐gp and Bcl‐xL. High levels of COX‐2, mPGES1, P‐gp, and Bcl‐xL expression were found in intestinal‐type gastric cancer samples. In conclusion, H pylori‐dependent induction of COX‐2 and mPGES1 is associated with enhanced production of P‐gp and Bcl‐xL that may contribute to gastric tumourigenesis and resistance to therapy. Copyright

Inhibitory effects of anti-CXCR4 antibodies on human colon cancer cells.

Background: CXCR4, the chemokine receptor for CXCL12, has recently been involved in the metastatic process of several neoplasms. Materials and methods: The expression of CXCR4 was evaluated by immunohistochemistry of colorectal tissue samples and by flow cytometry on Caco2, GEO, SW480, SW48, Lovo and SW620 human colon carcinoma cell lines. Correlations with pathological characteristics of the specimens were analysed with chi-square test. To verify the functional status of CXCR4, cell lines were tested in adhesion, migration, and proliferation assays. Results: We studied the expression of CXCR4 in 88 human colorectal tissues and we found that CXCR4 was expressed in >10% of epithelial cells in 50% of normal mucosae (7/14), in 55% of polyps (29/53), in all of carcinomas (16/16) and hepatic metastasis (5/5). Notably, CXCR4 was significantly over-expressed in cancerous lesions (carcinomas and metastasis) compared to non-cancerous lesions (normal mucosa and polyps) (P=0.003) and in adenomatous polyps versus hyperplastic polyps (P=0.009). The diameter of a polyp was also significantly associated with CXCR4 expression (P=0.031). SW480, SW48 and SW620 cell lines showed the highest levels of CXCR4 (60–80% of positive cells). Adhesion, migration, and proliferation increased in response to the CXCL12 chemokine. These effects were abrogated by the addition of anti-CXCR4 antibodies. Further, CXCL12 activated ERK1/2 in SW480 cells. Conclusions: These data suggest that CXCR4 might play a role in colon cancer cell properties and that anti-CXCR4 antibodies could have therapeutic effects against colorectal cancer.

Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular carcinoma associated with hepatitis B and hepatitis C virus infections

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Hepatocarcinogenesis is a multistep process mainly associated with persistent infection with hepatitis B (HBV) or C (HCV) viruses and always involving the accumulation of genetic alterations over decades of chronic liver disease. Mutations in TP53 and CTNNB1 genes are considered the cancer drivers for HCC development with variable frequencies depending on the etiology. Here we present a comprehensive review evaluating somatic mutations in TP53 and CTNNB1 genes in HBV- and HCV-related HCCs. Moreover, we report the mutational analysis of TP53 (exons 4-9) and CTNNB1 (exon 3) as well as PIK3CA (exon 9) genes in HCC from Southern Italy. The overall mutation frequency of TP53 and CTNNB1 was 33.3%, while hotspot variations in PIK3CA were completely absent. CTNNB1 mutations were significantly associated with young age (P=0.019) and moderately/poorly differentiated HCV-related HCC (P=0.015). The extended analysis of genetic alterations will help to identify molecular markers for liver cancer prevention, diagnosis and treatment of HBV and HCV-associated liver cancer.

Analysis of APAF-1 expression in human cutaneous melanoma progression.

Abstract: APAF‐1 plays a pivotal role in mitochondria‐dependent apoptosis, binding to cytochrome c and favoring activation of caspase‐9. It has been shown that epigenetic silencing of the APAF‐1 gene is a common event in several metastatic melanoma cells in vitro. We determined, by Western blot, variation in the level of expression of APAF‐1 in several human melanoma cell lines and, by immunohistochemistry, in a group of 106 histological samples including benign and malignant melanocytic lesions. We observed APAF‐1 down‐regulation or loss of expression in two metastatic melanoma cell lines, compared to primary melanoma cell lines. The immunohistochemical analysis revealed a significant difference in APAF‐1 staining between nevi and melanomas. In addition, we found a significant negative correlation between APAF‐1 expression level and tumor thickness and between primary melanomas and metastases. We conclude that loss of APAF‐1 expression can be considered as an indicator of malignant transformation in melanoma.

Extra-mitochondrial localisation of frataxin and its association with IscU1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2.

Friedreichs ataxia is a recessive neurodegenerative disease due to insufficient expression of the mitochondrial protein frataxin. Although it has been shown that frataxin is involved in the control of intracellular iron metabolism, by interfering with the mitochondrial biosynthesis of proteins with iron/sulphur (Fe/S) clusters its role has not been well established. We studied frataxin protein and mRNA expression and localisation during cellular differentiation. We used the human colon adenocarcinoma cell line Caco-2, as it is considered a good model for intestinal epithelial differentiation and the study of intestinal iron metabolism. Here we report that the protein, but not the mRNA frataxin levels, increase during the enterocyte-like differentiation of Caco-2 cells, as well as in in-vivo-differentiated enterocytes at the upper half of the crypt-villus axis. Furthermore, subcellular fractionation and double immunostaining, followed by confocal analysis, reveal that frataxin localisation changes during Caco-2 cell differentiation. In particular, we found an extramitochondrial localisation of frataxin in differentiated cells. Finally, we demonstrate a physical interaction between extramitochondrial frataxin and IscU1, a cytoplasmic isoform of the human Fe/S cluster assembly machinery. Based on our data, we postulate that frataxin could be involved in the biosynthesis of iron-sulphur proteins not only within the mitochondria, but also in the extramitochondrial compartment. These findings might be of relevance for the understanding of both the pathogenesis of Friedreichs ataxia and the basic mechanism of Fe/S cluster biosynthesis.

CD133 and CD44 Cell surface markers do not identify cancer stem cells in primary human gastric tumors

Emerging evidence suggests that tumors contain and are driven by a cellular component that displays stem cell properties, the so‐called cancer stem cells (CSCs). CSCs have been identified in several solid human cancers; however, there are no data about CSCs in primary human gastric cancer (GC). By using CD133 and CD44 cell surface markers we investigated whether primary human GCs contain a cell subset expressing stem‐like properties and whether this subpopulation has tumor‐initiating properties in xenograft transplantation experiments. We examined tissues from 44 patients who underwent gastrectomy for primary GC. The tumorigenicity of the cells separated by flow cytometry using CD133 and CD44 surface markers was tested by subcutaneous or intraperitoneum injection in NOD/SCID and nude mice. GCs included in the study were intestinal in 34 cases and diffuse in 10 cases. All samples contained surface marker‐positive cells: CD133+ mean percentage 10.6% and CD133+/CD44+ mean percentage 27.7%, irrespective of cancer phenotype or grade of differentiation. Purified CD133+ and CD133+/CD44+ cells, obtained in sufficient number only in 12 intestinal type GC cases, failed to reproduce cancer in two mice models. However, the unseparated cells produced glandular‐like structures in 70% of the mice inoculated. In conclusion, although CD133+ and CD133+/CD44+ were detectable in human primary GCs, they neither expressed stem‐like properties nor exhibited tumor‐initiating properties in xenograft transplantation experiments. J. Cell. Physiol. 227: 2686–2693, 2012.