Mariacarla Andreozzi

Human and Mouse VEGFA-Amplified Hepatocellular Carcinomas Are Highly Sensitive to Sorafenib Treatment

UNLABELLED 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 biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte 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 with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs. SIGNIFICANCE Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib-the first-line treatment of advanced HCC-which has an overall moderate therapeutic efficacy.

High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth.

We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth‐promoting role in cell biology. NRBP1 interacts directly with TSC‐22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA).

VEGFA gene locus analysis across 80 human tumour types reveals gene amplification in several neoplastic entities

BackgroundAngiogenesis 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.AimsThis 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).ResultsWe 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.ConclusionAlbeit 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.

HMGA1 Expression in Human Hepatocellular Carcinoma Correlates with Poor Prognosis and Promotes Tumor Growth and Migration in in vitro Models

BACKGROUND: HMGA1 is a non-histone nuclear protein that regulates cellular proliferation, invasion and apoptosis and is overexpressed in many carcinomas. In this study we sought to explore the expression of HMGA1 in HCCs and cirrhotic tissues, and its effect in in vitro models. METHODS: We evaluated HMGA1 expression using gene expression microarrays (59 HCCs, of which 37 were matched with their corresponding cirrhotic tissue and 5 normal liver donors) and tissue microarray (192 HCCs, 108 cirrhotic tissues and 79 normal liver samples). HMGA1 expression was correlated with clinicopathologic features and patient outcome. Four liver cancer cell lines with stable induced or knockdown expression of HMGA1 were characterized using in vitro assays, including proliferation, migration and anchorage-independent growth. RESULTS: HMGA1 expression increased monotonically from normal liver tissues to cirrhotic tissue to HCC (P < .01) and was associated with Edmondson grade (P < .01). Overall, 51% and 42% of HCCs and cirrhotic tissues expressed HMGA1, respectively. Patients with HMGA1-positive HCCs had earlier disease progression and worse overall survival. Forced expression of HMGA1 in liver cancer models resulted in increased cell growth and migration, and vice versa. Soft agar assay showed that forced expression of HMGA1 led to increased foci formation, suggesting an oncogenic role of HMGA1 in hepatocarcinogenesis. CONCLUSIONS: HMGA1 is frequently expressed in cirrhotic tissues and HCCs and its expression is associated with high Edmondson grade and worse prognosis in HCC. Our results suggest that HMGA1 may act as oncogenic driver of progression, implicating it in tumor growth and migration potential in liver carcinogenesis.

SH2D4A is frequently downregulated in hepatocellular carcinoma and cirrhotic nodules

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.

Vascular endothelial growth factor A amplification in colorectal cancer is associated with reduced M1 and M2 macrophages and diminished PD-1-expressing lymphocytes

VEGFA is an angiogenic factor secreted by tumors, in particular those with VEGFA amplification, as well as by macrophages and lymphocytes in the tumor microenvironment. Here we sought to define the presence of M1/M2 macrophages, PD-1-positive lymphocytes and PD-L1 tumoral and stromal expression in colorectal cancers harboring VEGFA amplification or chromosome 6 polysomy. 38 CRCs of which 13 harbored VEGFA amplification, 6 with Chr6 polysomy and 19 with neutral VEGFA copy number were assessed by immunohistochemistry for CD68 (marker for M1/M2 macrophages), CD163 (M2 macrophages), programmed death 1(PD-1)- tumor infiltrating and stromal lymphocytes as well as tumoral and stromal PD-1 ligand (PD-L1) expression. CRCs with VEGFA amplification or Chr6 polysomy were associated with decreased M1/M2 macrophages, reduced PD-1-expressing lymphocyte infiltration, as well as reduced stromal expression of PD-L1 at the tumor front. Compared to intermediate-grade CRCs, high-grade CRCs were associated with increased M1/M2 macrophages and increased tumoral expression of PD-L1. Our results suggest that VEGFA amplification or Chr6 polysomy is associated with an altered tumor immune microenvironment.

Abstract 4685: SH2D4A is frequently downregulated in hepatocellular carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. The lack of effective therapeutic options combined with an increasing incidence rate in the past 20 years calls for the identification of early stage HCC molecular markers. SH2D4A maps to human chromosome 8p21.3 and encodes for SH(2)A. The chromosomal region containing SH2D4A has been shown to be frequently lost in colorectal, lung and HCC cancers. Using a combination of gene expression, protein level and clinical data, our study aimed to investigate the SH2D4A involvement in HCC pathogenesis. Transcriptome analysis (Affymetrix) performed on 37 HCC samples (matched with their corresponding non-neoplastic liver parenchyma) and 5 normal liver donors revealed that SH2D4A is downregulated in HCC. These results were validated by qPCR in fresh frozen tissues [25 out of 37 samples (67.6%) showed SH2D4A downregulation; p<0.026]. Furthermore, the combination of qPCR data and immunohistochemistry staining performed on corresponding formalin-fixed paraffin embedded (FFPE) needle biopsies demonstrated a direct correlation between the SH2D4A mRNA expression and SH(2)A protein levels. Additional data obtained from the analysis of a tissue microarray (TMA) containing 336 informative specimens confirmed that SH(2)A protein levels are frequently reduced in HCC and additionally in cirrhosis nodules compared to normal liver samples. To conclude, our study revealed that SH2D4A is frequently downregulated in HCC samples thus corroborating its putative role as tumor suppressor gene. In addition, we provide new evidence for SH2D4A involvement in HCC pathogenesis demonstrating for the first time its deregulation also in cirrhotic nodules. Citation Format: Mariacarla Andreozzi, Luca Quagliata, Michal Kovac, Luigi Tornillo, Zuzanna Makowska, Marcus Heim, Karl Heinimann, Salvatore Piscuoglio, Luigi Maria Terracciano. SH2D4A is frequently downregulated in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4685. doi:10.1158/1538-7445.AM2013-4685 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5295: Detection of VEGFA gene amplification in different neoplastic entities using four different technologies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background The VEGFA protein plays an important role in the induction of angiogenesis during tumor initiation and progression. The aims of the study were to investigate the presence of VEGFA gene amplification by FISH analysis on a human multi-tumor tissue microarray (MTMA) and to validate these findings on an independent human multi-tumor tissue in mouse implanted microarray (MTMA) by aCGH, SNP6 microarrays, FISH analysis and real-time PCR. Materials and Methods The VEGFA gene status was evaluated on a human MTMA (n=2,957 tissue samples) including 132 tumor entities and 31 normal tissue types. Furthermore, we reconfirmed the presence of VEGFA gene amplification as detected by FISH analysis on a second independent MTMA including 195 different tissue samples from nude mice xenograft models of 24 different human tumor types using three additional genomic techniques: array-CGH, SNP6 microarrays and real-time PCR. Results VEGFA gene amplification was detected across different tumor types. The following incidences were observed: 5.3% in colorectal carcinoma, 3.2% in endometrial carcinoma, 2.8% in gallbladder adenocarcinoma, 4.2 % in renal carcinoma, 1.5% in hepatocellular carcinoma, 4.5% in pancreas carcinoma and 4.8% in stomach carcinoma samples. Additionally, some tumors were characterized by a high polysomy. In the second independent cohort MTMA VEGFA gene amplification was detected by FISH analysis in four samples, including one non-small cell lung cancer, one sarcoma, one gastric and and breast cancer, previously classified as amplified by aCGH (100%) and SNP6 microarrays. The latter technique also revealed one “supplementary amplified sample”. All results correlated with real-time PCR (p<0.05). Conclusions We detected VEGFA gene amplification in different tumor types, on a human MTMA and we confirmed our findings on an independent MTMA of human tumors implanted in mice. We demonstrated the occurrence of VEGFA gene amplification in several tumor types with an incidence of up to 5% and also we demonstrated the feasibility of four technologies for reliable VEGFA gene copy number detection. Comparison of the four technologies revealed high concordance (p<0.05). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5295. doi:1538-7445.AM2012-5295

Abstract 5142: VEGFA gene amplification and protein expression in breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression. Methods: VEGFA gene amplification was evaluated by fluorescence in situ hybridization (FISH) on a multitumor tissue microarray (MTMA) comprising 3417 tissue samples from more than 100 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence immunoassay. In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers. Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 70) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p<0.0001). Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5142. doi:10.1158/1538-7445.AM2011-5142

Abstract 3815: Stage I non-small cell lung cancer expressing SOX2 show more recurrence in elderly male patients with non-adenocarcinoma histology

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Around 30% of patients with early-stage non-small cell lung cancer (NSCLC) relapse after surgery. Therefore, new prognostic and predictive molecular markers are needed to identify patients who could benefit from adjuvant treatment. SOX2 (sex determining region Y-box 2) at the gene locus 3q26.33 is regarded as an oncogene involved in carcinogenesis of squamous cell lung carcinoma. Nevertheless, comprehensive data on SOX2 gene status and expression as well as its prognostic relevance in early-stage NSCLC are not yet available. The aim of the present study was to investigate the prevalence and the prognostic significance of SOX2 gene status and expression in a large series of stage I NSCLC patients. Methods: A tissue micro array with 568 paraffin-embedded stage I NSCLC with comprehensive histopathological and clinical data, including follow-up on overall and tumor-specific survival was analyzed. SOX2 gene status was determined by fluorescent in situ hybridization (FISH) using direct-labeled BAC clones (SOX2: RP11-938O9; reference probe (RP): RP11-286G5). SOX2 gene status was recorded as follows: Amplification (ratio SOX2/RP ≥2.2), polysomy (more than 4 signals in the CEP or the gene probe) and normal (ratio: 0.8-1.8). Semi-quantitative expression of SOX2 protein was determined by immunohistochemistry (IHC). Any nuclear reaction of tumor cells was regarded as a positive result. FISH and IHC data were correlated with clinicopathological features and overall survival. Results: An increased SOX2 gene number (amplification or polysomy) was observed in 4% of NSCLC (17/429). 94% of NSCLC (16/17) with an increased SOX2 gene number were observed in squamous- and large cell carcinomas (non-adenocarcionomas). Using logistic regression analysis an increased SOX2 gene number was significantly (p 50years; p=0.001) and male (p>0.001) patients. Elderly male patients with non-adenocarcionoma histology and a smoking history expressing SOX2 had a tendency toward more recurrence (p=0.06). Conclusions: An increased SOX2 gene number is rare in stage I NSCLC and it is mostly detected in squamous and large cell carcinomas. SOX2 protein expression stratifies a subgroup of NSCLC from elderly patients with a smoking history and non-adenocarcinoma histology. SOX2 two could become a potential therapeutic target molecule in a subgroup of patients with early stage NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3815. doi:10.1158/1538-7445.AM2011-3815