Paired Liver Metastases Research Articles

Differential Protein Expression in Small Intestinal Neuroendocrine Tumors and Liver Metastases.

Small intestinal neuroendocrine tumors (SI-NETs) are often detected after they have become metastatic. Using a novel protein array, we identified pathways important in SI-NET metastasis development in surgically resected patients. Paired primary tumors and liver metastases from 25 patients undergoing surgical resection for metastatic SI-NETs were harvested. Extracted proteins were separated by sodium dodecyl sulfate gel and multiplex immunoblots were performed with 136 antibodies. Significant Analysis of Microarray was used to select for differentially expressed proteins. A tissue microarray was constructed from 27 archived specimens and stained by immunohistochemistry. Comparing primary SI-NETs with matched normal small-bowel mucosa, 9 proteins were upregulated and cyclin E was downregulated. The SI-NET liver metastases demonstrated upregulation of P-ERK and p27 but downregulation of CDK2 and CDC25B. When comparing primary SI-NET with their paired liver metastases, cyclin E demonstrated a significant upregulation in the liver metastasis. Tissue microarray demonstrated higher p38 expression and lower Cdc 25b expression in SI-NETs versus liver metastases and confirmed higher expression of p27 in liver metastases versus normal liver. Few studies have compared protein expression in paired primary and metastatic SI-NETs. Our findings reveal changes in a limited number of proteins, suggesting that these may be targets for therapy.

Clinical impact of c-MET expression and genetic mutational status in colorectal cancer patients after liver resection.

c-MET is implicated in the pathogenesis and growth of a wide variety of human malignancies, including colorectal cancer (CRC). The aim of the present study was to clarify the association between c-MET expression and tumor recurrence in CRC patients after curative liver resection, and to evaluate concordance in c-MET expression and various mutations of KRAS, BRAF and PIK3CA between primary CRC and paired liver metastases. A cohort of patients was tested for c-MET immunoreactivity (i.e. immunohistochemistry [IHC]) and KRAS, BRAF and PIK3CA mutations. Analyses were performed both on primary tumors and paired liver metastases, and the association between IHC and mutations results were assessed. A total of 108 patients were eligible. A total of 53% of patients underwent simultaneous resection of primary tumors and metastases, and the others underwent metachronous resection. Levels of concordance between primary tumors and metastases were 65.7%, 87.7%, 100% and 95.2% for c-MET, KRAS, BRAF and PIK3CA, respectively. High levels of c-MET expression (c-MET-high) in the primary tumors were observed in 52% of patients. Relapse-free survival was significantly shorter for patients with c-MET-high primary tumors (9.7 months) than for those with c-MET-low primary tumors (21.1 months) (P = 0.013). These results suggest that a high level of genetic concordance in KRAS, BRAF and PIK3CA between primary tumors and liver metastases, and c-MET-high in the primary tumors were associated with shorter relapse-free survival after hepatic metastasectomy.

Concordance of HER2 and its related molecules between primary and paired liver metastatic sites in gastric cancer.

4108 Background: Trastuzumab is the 1st molecular targeting drug in HER2-positive advanced gastric cancer that has been shown to confer overall survival benefit adding to chemotherapy. In breast cancer it has been already used long time, it is known that there are the discordance of its target; HER2 between primary and metastatic site. Although molecular targeting drugs as Trastuzumab are expected to control the metastatic disease, molecular status is usually evaluated in the primary site because metastatic sites are difficult to biopsy. In this study we attempted to compare HER2 and its related molecular status, which have interaction each together, between primary and paired liver metastatic sites in gastric cancer. Methods: Total 58 consecutive cases with gastric cancer who underwent surgical resection of primary site and synchronous or metachronous liver metastasis were examined. HER2, EGFR, c-MET and IGF-1R status were evaluated by immunohistochemistry(IHC) in primary and paired liver metastasis. HER2 expression by IHC using HercepTest (DAKO) were assessed according to Gastric Cancer Scoring System. On the other molecular expression by IHC, positive expression was defined as 25% or more staining with intensity 2 or 3+. We analyzed the concordance of their expression in both sites. Results: The patient cohort consists predominantly of male (78%), with Lauren’s diffuse (37%) and intestinal tumors (62%). Fifty three percent of cases were synchronous liver metastasis. The positive rate of primary and paired liver metastatic sites were 10.3%, 8.6% in HER2, 1.7%, 5.1% in EGFR, 44.8%, 31.0% in c-MET and 31.0%, 29.3% in IGF-1R. The concordance between primary and paired liver metastatic sites were 91.3%, 93.1%, 75.8%, and 70.6%, respectively. Conclusions: Our data suggested that in HER2 and EGFR the concordance between primary and metastatic site were high, other hands the concordance of c-MET and IGF-1R were relatively low. It might be necessary to rebiopsy to estimate the expression of c-MET and IGF-1R in gastric cancer.

The Insulin Receptor Substrate 1 (Irs1) in Intestinal Epithelial Differentiation and in Colorectal Cancer

Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.

Abstract 4529: Prognostic implications of TSPO in human colorectal cancer

Abstract Translocator protein (TSPO), previously referred to as peripheral benzodiazepine receptor, is an 18kDa outer mitochondrial membrane protein involved in a number of cellular processes including steroid synthesis, cholesterol metabolism, proliferation, and apoptosis. Elevated TSPO expression has been linked with disease progression, diminished survival, and aggressiveness in a number of cancers. Studies conducted in our laboratory have demonstrated that positron emission tomography (PET) imaging with TSPO targeted probes can be used to non-invasively quantify TSPO levels in tumors. These imaging studies suggest that TSPO ligand PET may be a valuable clinical cancer imaging biomarker. In the current studies, TSPO expression in human colon cancer and paired liver metastases was evaluated using tissue microarrays. These arrays were constructed using 99 primary colon cancers and 95 matched primary colon cancers and liver metastases from Vanderbilt patients with known clinical outcomes. Among these, we found that nearly 2/3 of the colon cancers were positive for TSPO expression (64%). Similar to previous studies in other organ sites, elevated TSPO expression was significantly associated with higher tumor grade in the Vanderbilt cohort of CRC patients. Among patients with grade 2 or grade 3 tumors, an individual with elevated TSPO expression was 3 times more likely to have a grade 3 tumor than a grade 2 tumor. In comparing matched primary colon cancers and associated liver metastases, we found a strong concordance between TSPO levels and localization across matched specimens. No correlation was observed between TSPO expression and Ki67 or pEGFR levels suggesting the potentially unique information provided by the measurement of TSPO levels. In conclusion, clinically relevant prognostic information can be obtained by the measurement of TSPO expression in colorectal cancer. Thus, TSPO targeted PET may be a promising-non invasive biomarker in this setting. 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 4529. doi:1538-7445.AM2012-4529

DNA Methylation Profiles of Primary Colorectal Carcinoma and Matched Liver Metastasis

BackgroundThe contribution of DNA methylation to the metastatic process in colorectal cancers (CRCs) is unclear.MethodsWe evaluated the methylation status of 13 genes (MINT1, MINT2, MINT31, MLH1, p16, p14, TIMP3, CDH1, CDH13, THBS1, MGMT, HPP1 and ERα) by bisulfite-pyrosequencing in 79 CRCs comprising 36 CRCs without liver metastasis and 43 CRCs with liver metastasis, including 16 paired primary CRCs and liver metastasis. We also performed methylated CpG island amplification microarrays (MCAM) in three paired primary and metastatic cancers.ResultsMethylation of p14, TIMP3 and HPP1 in primary CRCs progressively decreased from absence to presence of liver metastasis (13.1% vs. 4.3%; 14.8% vs. 3.7%; 43.9% vs. 35.8%, respectively) (P<.05). When paired primary and metastatic tumors were compared, only MGMT methylation was significantly higher in metastatic cancers (27.4% vs. 13.4%, P = .013), and this difference was due to an increase in methylation density rather than frequency in the majority of cases. MCAM showed an average 7.4% increase in DNA methylated genes in the metastatic samples. The numbers of differentially hypermethylated genes in the liver metastases increased with increasing time between resection of the primary and resection of the liver metastasis. Bisulfite-pyrosequencing validation in 12 paired samples showed that most of these increases were not conserved, and could be explained by differences in methylation density rather than frequency.ConclusionsMost DNA methylation differences between primary CRCs and matched liver metastasis are due to random variation and an increase in DNA methylation density rather than de-novo inactivation and silencing. Thus, DNA methylation changes occur for the most part before progression to liver metastasis.

Heterogeneity of Kras status in primary colorectal cancer and corresponding liver metastases.

e14037 Background: It is well known that Kras mutation status is a definitive marker for the treatment of colorectal cancer when anti-EGFR antibody agents are used. Several previous reports demonstrated that the Kras status in primary tumor and paired metastases were well concordant, although some samples showed mismatch status between them. Recent report assumed that the one of the reasons of this mismatch was due to intratumoral heterogeneity of Kras mutation status. Methods: Ninety colorectal cancer patients with primary tumor and liver metastases (76 were synchronous metastases and 14 were metachronous) were evaluated. Cancer cells of primary tumor and paired liver metastases were selectively dissected from formalin-fixed paraffin embedded samples with using Laser-captured microdissection. The Kras sequences of codon 12, 13 were analyzed by direct sequencing. Results: The concordance rate of Kras mutation status between primary tumor and paired liver metastases were 88.2% in synchronous metastases, and 100% in metachronous. Nine patients showed different Kras status between primary and metastases. Twelve patients had received liver resection for twice, 2 patients (17%) showed different Kras status between the samples from first liver resection and second resection. We assumed this discrepancy was due to intratumoral heterogeneity, four different areas of primary tumor were dissected; two from invasion front areas and other two from surface areas of each tumor in 9 patients who showed different Kras status between primary and metastases, and Kras status were compared. Seven out of 9 patients showed at least one area of mismatched Kras status, suggesting the existence of intratumoral heterogeneities. Conclusions: The Kras status was matched between primary and paired liver metastases in most of the patients, although some patients showed mismatch status. Intratumoral heterogeneity is assumed to be one of the reasons of this mismatch.

Dynamics of cancer cell subpopulations in primary and metastatic colorectal tumors

Intratumor heterogeneity—heterogeneity of cancer cells within a single tumor—is considered one of the most problematic factors of treatment. Genetic heterogeneity, such as in somatic mutations and chromosome aberrations, is a common characteristic of human solid tumors and is probably the basis of biological heterogeneity. Using mutations in APC, TP53 and KRAS as markers to identify distinct colorectal cancer subpopulations, we analyzed a total of 42 primary colorectal cancer tissues and six paired liver metastases with multipoint microsampling, which enabled analysis of mutation patterns and allelic imbalances with a resolution of 0.01 mm2 (about 200 cells). There was usually more than one subpopulation in each primary tumor. Only two of 15 (13.3%) cases with three gene mutations and eight of 27 (29.6%) cases with two gene mutations had a single subpopulation. Cells with mutations in all of the examined genes usually constituted the major population. Multipoint microsampling of six primary and metastatic tumor pairs revealed that the majority of discrepancies in mutation patterns found with the bulk tissue analysis were due to loss of subpopulations in the metastatic tissues. In addition, multipoint microsampling uncovered substantial changes in subpopulations that were not detected with bulk tissue analysis. Specifically, the proportion of KRAS mutation-negative subpopulations increased in the metastatic tumors of four cases. Because KRAS mutation status is linked to cetuximab/panitumumab efficacy, subpopulation dynamics could lead to differences in response to cetuximab/panitumumab in primary versus metastatic tumors.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-011-9381-0) contains supplementary material, which is available to authorized users.

Genetic heterogeneity in lung and colorectal carcinoma as revealed by microsatellite analysis in plasma or tumor tissue DNA.

Determination of tumor clonality has implications for molecular characterization and the optimal treatment of cancer. Allelotyping allows detection of the two alleles, maternal and paternal, and provides additional information regarding clonal genetic defects. The presence of allelic imbalances (AI) in tumors is a general event, but is not necessary at the same allele (alternative AI). The authors' goal was to determine whether the presence of alternative AI (AA) was a marker of heterogeneity and prognosis. To further analyze the heterogeneity of lung tumors, tumor DNA released in the plasma was compared with primary tumor DNA from 24 lung carcinoma patients. The comparison was performed by allelotyping using 12 microsatellites targeting 9 chromosomal regions, taking in each case leukocyte DNA as reference. To extend and confirm these observations, 26 primary colorectal carcinomas with paired synchronous liver metastasis were analyzed using an enlarged panel of 33 microsatellites. AA were observed in 40% (20 of 50) of all patients, in 25% (6 of 24) of lung carcinoma patients but at a higher level, and in 54% (14 of 26) of colorectal carcinoma patients. They affected different chromosome localizations and each tumor stage. In both types of cancer, patients with AA had a higher AI mean frequency in their primary tumor. Detection of AA is an original marker of heterogeneous tumors, demonstrating that independent events occurred on specific genetic sites required for cancer progression.