[Show abstract][Hide abstract] ABSTRACT: Colorectal cancer (CRC) infiltration by CD16+ myeloid cells correlates with improved prognosis. We addressed mechanistic clues, and gene and protein expression of cytokines potentially associated with macrophage polarization.
GM-CSF or M-CSF stimulated peripheral blood CD14+ cells from healthy donors were co-cultured with CRC cells. Tumor cell proliferation was assessed by 3H-Thymidine incorporation. Expression of cytokine (CK) genes in CRC and autologous healthy mucosa (HM) was tested by quantitative, real-time PCR. A tumor microarray (TMA) including >1200 CRC specimens was stained with GM-CSF and M-CSF specific antibodies. Clinic-pathological features and overall survival were analyzed.
GM-CSF induced CD16 expression in 66±8% of monocytes, as compared to 28±1% in cells stimulated by M-CSF (P=0.011). GM-CSF but not M-CSF stimulated macrophages significantly (P<0.02) inhibited CRC cell proliferation. GM-CSF gene was expressed to significantly (n=45, P<0.0001) higher extents in CRC than in HM whereas M-CSF gene expression was similar in HM and CRC. Accordingly, IL-1β and IL-23 genes, typically expressed by M1 macrophages, were expressed to significantly (P<0.001) higher extents in CRC than in HM. TMA staining revealed that GM-CSF production by tumor cells is associated with lower T stage (p=0.02), "pushing" growth pattern (P=0.004) and significantly (P=0.0002) longer survival in mismatch-repair proficient (MMRp) CRC. Favorable prognostic effect of GM-CSF production by CRC cells was confirmed by multivariate analysis and was independent from CD16+ and CD8+ cell CRC infiltration. M-CSF expression had no significant prognostic relevance.
GM-CSF production by tumor cells is an independent favorable prognostic factor in CRC.
Clinical Cancer Research 04/2014; · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Death rates from hepatocellular carcinoma (HCC) are steadily increasing yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biological characteristics. Unlike common tumor amplifications, this one appears to work via heterotypic paracrine interactions: stromal VEGF receptors (VEGFRs), responding to tumor VEGF-A, produce hepatoycte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib - the first-line drug in advanced HCC - targets multiple kinases including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients withVEGFA amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A blocking drugs.
[Show abstract][Hide abstract] ABSTRACT: The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC) tumors, its association with the clinical course of the disease, and the underlying mechanism(s). Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ) induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1β (IL-1β) production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1β production by monocytes.
Neoplasia (New York, N.Y.) 01/2014; 16(1):31-42. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. The lack of effective therapeutic options for advanced stage HCCs combined with an increasing incidence rate calls for the identification of early stage HCC molecular markers. SH2 Domain Containing 4A (SH2D4A) gene maps to human chromosome 8p21.3 and encodes for SH(2)A. The chromosomal region containing SH2D4A is frequently lost in colorectal, lung and HCC cancers. Our study aimed to investigate SH2D4A involvement in HCC pathogenesis combining mRNA expression, protein and clinical data. Transcriptome analysis performed on 37 HCC needle biopsies (matched with their corresponding non-neoplastic parenchyma) and five normal liver donor samples revealed that SH2D4A is downregulated in HCC. Results were confirmed by quantitative real-time-polymerase chain reaction (qRT-PCR), 25 out of 37 (67.6%) fresh frozen samples showed SH2D4A downregulation (p=0.026). Furthermore, combining qRT-PCR and immunohistochemistry data we demonstrated a direct correlation between SH2D4A mRNA and SH(2)A protein levels. The analysis of a tissue microarray (TMA) containing 336 specimens confirmed that SH(2)A is frequently reduced in HCC (56.8%) as well as in cirrhotic nodules (50.5%) compared to normal liver samples (31.1%). To conclude, our study revealed that SH2D4A is frequently downregulated in HCC samples thus corroborating its putative role as a tumour suppressor gene. In addition, we provide new evidence for SH2D4A involvement in HCC pathogenesis demonstrating for the first time its deregulation in cirrhotic nodules.
European journal of cancer (Oxford, England: 1990) 12/2013; · 4.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis plays a pivotal role in neoplastic growth and metastasis formation. Vascular endothelial growth factor A (VEGFA) is a major player in physiological and tumour-induced angiogenesis and numerous human tumours have been show to overexpress VEGFA. Moreover increased VEGFA gene expression has been found frequently to correlate with tumour progression, recurrences and survival. Interestingly, several studies have demonstrated that gene amplification may result in protein overexpression and that amplification of the therapeutics' target gene can serve as an excellent predictive marker (i.e. HER2 and trastuzumab). However the impact of VEGFA gene amplification has been only recently assessed for some cancer types such as osteosarcoma, colorectal, breast and liver cancer.
This study aimed to assess VEGFA gene amplification status using fluorescent in situ hybridization (FISH) in a large cohort of different tumour entities. Thus, we investigated the incidence of VEGFA amplification using a multi-tumour tissue microarray (TMA) containing 2,837 evaluable specimens from 80 different tumour entities and 31 normal tissue types. Moreover, we validated FISH analysis as reference method to evaluate VEGFA gene status by comparing it to comparative genomic hybridization (CGH).
We observed that VEGFA locus amplification and/or polysomy represented a small but regularly detected population in several tumour entities while was not present in normal tissues. VEGFA gene alterations were predominantly observed in hepatocarcinomas, adenocarcinomas of the pancreas and intestine, large cell carcinoma of the lung and in endometrium serous carcinoma. Furthermore our data demonstrated that VEGFA detection by FISH provided highly comparable results to those generated by CGH.
Albeit with low percentage, VEGFA amplification is commonly observed across several tumour entities. Furthermore, our results demonstrated that FISH test could be used as a reliable diagnostic tool to evaluate VEGFA gene status in human specimens.
[Show abstract][Hide abstract] ABSTRACT: The prognostic relevance of innate immune cells infiltrating colorectal carcinoma lesions is highly debated. By evaluating the expression of myeloperoxidase (MPO) as a marker of neutrophil granulocytes in a large cohort of colorectal carcinoma specimens, we have observed that robust tumor-infiltration by MPO(+) cells correlates with improved patient survival independently of other histopathological parameters, including disease stage.
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death. Despite the advances in diagnosis and management of HCC, the biology of this tumor remains poorly understood. Recent evidence highlighted long non-coding RNAs (lncRNAs) as crucial determinants of HCC development. In this study, we report the lncRNA HOTTIP as significantly up-regulated in HCC specimens. HOTTIP gene is located in physical contiguity with HOXA13 and directly controls the HOXA locus gene expression via interaction with the WDR5 / MLL complex. HOX genes encode transcription factors regulating embryonic development and cell fate. We previously described HOX genes deregulation to be involved in hepatocarcinogenesis. Indeed, we observed the marked up-regulation of HOXA13 in HCC. Here, by correlating clinico-pathological and expression data, we demonstrate that the levels of HOTTIP and HOXA13 are associated with HCC patients' clinical progression and predict disease outcome. In contrast to the majority of similar studies, our data are obtained from snap-frozen needle HCC biopsies (n=52) matched with their non-neoplastic counterparts collected from patients that had not yet received any HCC-tailored therapeutic treatments at the time of biopsy. In addition, taking advantage of gain and loss of function experiments in liver cancer-derived cell lines (HuH-6 and HuH-7), we uncover a novel bidirectional regulatory loop between HOTTIP / HOXA13. Conclusion: our study highlights the key role of HOTTIP and HOXA13 in HCC development by associating their expression to metastasis and survival in HCC patients, provides novel insights on the function of lncRNA-driven hepatocarcinogenesis and paves the way for further investigation about the possible role of HOTTIP as predictive biomarker of HCC. (Hepatology 2013;).
[Show abstract][Hide abstract] ABSTRACT: Nodular regenerative hyperplasia (NRH) is a rare liver disease characterized by small regenerative nodules without fibrosis and can cause portal hypertension. Aetiology and pathogenesis of NRH remain unclear. We have recently shown that Notch1 knockout induces NRH with portal hypertension through vascular remodelling in mice. The aim of this study was to analyse histological and clinical data of NRH patients and to explore if the endothelial pathways identified in our NRH mouse model are also regulated in human NRH.
Patients were identified retrospectively from the pathology database. Clinical and laboratory patient data were retrieved. mRNA expression was measured in liver biopsies from a subset of NRH patients.
Diagnosis of NRH was confirmed in needle biopsies of 51 patients, including 31 patients with grade 1, 12 patients with grade 2 and 8 patients with grade 3 NRH. Grade 3 nodularity significantly correlated with the presence of portal hypertension: 50% of the patients with grade 3 NRH vs. 6.5% with grade 1 (P = 0.0105). mRNA expression analysis in liver biopsies from 14 NRH patients and in primary human sinusoidal endothelial cells revealed downregulation of identical genes as in the murine NRH model, which are implicated in vascular differentiation: Notch1, delta-like 4 (Dll4) and ephrinB2.
In this large NRH needle biopsy cohort, we demonstrated that advanced nodularity correlates with presence of portal hypertension. Downregulation of the endothelial signalling pathways Dll4/Notch1 and ephrinB2/EphB4 supports the hypothesis that human NRH is caused by a sinusoidal injury providing first insights into the molecular pathogenesis of this liver condition.
Liver international: official journal of the International Association for the Study of the Liver 06/2013; · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS: A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS: Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8+ lymphocytes (P=0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P=0.0001) and validation (P=0.03) sets. A similar trend (P=0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n=42) were found to be significantly associated (r=0.33, P=0.03). CONCLUSION: PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.
European journal of cancer (Oxford, England: 1990) 03/2013; · 4.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Congenital malignant melanoma (CMM) is a rare condition that is defined as malignant melanoma recognized at birth. CMM may develop in utero in one of three ways: (1) transmission by metastasis through the placenta from a mother with melanoma; (2) primary melanoma arising within a giant congenital melanocytic naevus (GCMN); (3) primary de novo cutaneous CMM arising in utero. CMM can be confused clinically and histologically with benign proliferative melanocytic lesions such as giant congenital nevi. We describe the case of a patient presenting a GCMN with proliferative nodules, clinically and dermoscopically resembling a CMM, demonstrating the importance of caution in making a diagnosis of MM and highlighting the possibility that benign lesions as GCMN can mimic a malignant melanoma in this age group.
Case reports in dermatological medicine. 01/2013; 2013:473635.
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG).
A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets.
MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO-.
High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC.
PLoS ONE 01/2013; 8(5):e64814. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acinic cell carcinoma is a rare breast tumour belonging to salivary gland-like tumours of the breast. They are "triple-negative" breast cancers even if their biological behaviour seems to be more favourable. Herein we present an acinic cell carcinoma arising on a background of typical and atypical microglandular adenosis in a 58-year-old woman, along with a review of the literature.
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) are the most common liver tumors and a leading cause for cancer-related death in men. Notch2regulates cellular differentiation in the developing and adult liver. While aberrant Notch2 signaling is implicated in various cancers, it is still unclear whether Notch2 regulates proliferation and differentiation in liver carcinogenesis and thereby contributes to HCC and CCC formation. Here, we investigated the oncogenic potential of constitutive Notch2 signaling in the liver. We show that liver-specific expression of the intracellular domain of Notch2 (N2ICD) in mice is sufficient to induce HCC formation and biliary hyperplasia. Specifically, constitutive N2ICD signaling in the liver leads to upregulation of pro-proliferative genes and proliferation of hepatocytes and biliary epithelial cells (BECs). Using the diethylnitrosamine (DEN) HCC carcinogenesis model, we further show that constitutive Notch2 signaling accelerates DEN-induced HCC formation. DEN-induced HCCs with constitutive Notch2 signaling (DEN(N2ICD) HCCs) exhibit a marked increase in size, proliferation and expression of pro-proliferative genes when compared to HCCs from DEN-induced control mice (DEN(ctrl) HCCs). Moreover, DEN(N2ICD) HCCs exhibit increased Sox9 mRNA levels and reduced Albumin and Alpha Fetoprotein mRNA levels, indicating that they are less differentiated than DEN(ctrl) HCCs. Additionally, DEN(N2ICD) mice develop large hepatic cysts, dysplasia of the biliary epithelium and eventually CCC. CCC formation in patients and DEN(N2ICD) mice is accompanied by re-expression of HNF4α, possibly indicating dedifferentiation of BECs. Conclusion: Our data establish an oncogenic role for constitutive Notch2 signaling in liver cancer development.(HEPATOLOGY 2012.).
[Show abstract][Hide abstract] ABSTRACT: Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response to treatment with pegylated interferon (pegIFN)-α and ribavirin. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with sustained virologic response rates greater than 90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy.
We analyzed IFN signaling and ISG expression in liver samples from patients with AHC, patients with CHC, and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets.
Expression levels of hundreds of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ-stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α-stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of suppresor of cytokine signaling 1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC.
Differences in expression of ISGs might account for the greater response of patients with AHC, compared with those with CHC, to treatment with pegIFN-α and ribavirin. Specifically, USP18 is up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in patients with AHC.
[Show abstract][Hide abstract] ABSTRACT: Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.
Archives of Oto-Rhino-Laryngology 05/2012; · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.
Diagnostic molecular pathology: the American journal of surgical pathology, part B 03/2012; 21(1):14-23. · 1.58 Impact Factor