Zhaoshi Zeng

Relationship of Gene Expression and Chromosomal Abnormalities in Colorectal Cancer

Several studies have verified the existence of multiple chromosomal abnormalities in colon cancer. However, the relationships between DNA copy number and gene expression have not been adequately explored nor globally monitored during the progression of the disease. In this work, three types of array-generated data (expression, single nucleotide polymorphism, and comparative genomic hybridization) were collected from a large set of colon cancer patients at various stages of the disease. Probes were annotated to specific chromosomal locations and coordinated alterations in DNA copy number and transcription levels were revealed at specific positions. We show that across many large regions of the genome, changes in expression level are correlated with alterations in DNA content. Often, large chromosomal segments, containing multiple genes, are transcriptionally affected in a coordinated way, and we show that the underlying mechanism is a corresponding change in DNA content. This implies that whereas specific chromosomal abnormalities may arise stochastically, the associated changes in expression of some or all of the affected genes are responsible for selecting cells bearing these abnormalities for clonal expansion. Indeed, particular chromosomal regions are frequently gained and overexpressed (e.g., 7p, 8q, 13q, and 20q) or lost and underexpressed (e.g., 1p, 4, 5q, 8p, 14q, 15q, and 18) in primary colon tumors, making it likely that these changes favor tumorigenicity. Furthermore, we show that these aberrations are absent in normal colon mucosa, appear in benign adenomas (albeit only in a small fraction of the samples), become more frequent as disease advances, and are found in the majority of metastatic samples.

Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

Comparative Genomic Analysis of Primary Versus Metastatic Colorectal Carcinomas

PURPOSE To compare the mutational and copy number profiles of primary and metastatic colorectal carcinomas (CRCs) using both unpaired and paired samples derived from primary and metastatic disease sites. PATIENTS AND METHODS We performed a multiplatform genomic analysis of 736 fresh frozen CRC tumors from 613 patients. The cohort included 84 patients in whom tumor tissue from both primary and metastatic sites was available and 31 patients with pairs of metastases. Tumors were analyzed for mutations in the KRAS, NRAS, BRAF, PIK3CA, and TP53 genes, with discordant results between paired samples further investigated by analyzing formalin-fixed, paraffin-embedded tissue and/or by 454 sequencing. Copy number aberrations in primary tumors and matched metastases were analyzed by comparative genomic hybridization (CGH). RESULTS TP53 mutations were more frequent in metastatic versus primary tumors (53.1% v 30.3%, respectively; P < .001), whereas BRAF mutations were significantly less frequent (1.9% v 7.7%, respectively; P = .01). The mutational status of the matched pairs was highly concordant (> 90% concordance for all five genes). Clonality analysis of array CGH data suggested that multiple CRC primary tumors or treatment-associated effects were likely etiologies for mutational and/or copy number profile differences between primary tumors and metastases. CONCLUSION For determining RAS, BRAF, and PIK3CA mutational status, genotyping of the primary CRC is sufficient for most patients. Biopsy of a metastatic site should be considered in patients with a history of multiple primary carcinomas and in the case of TP53 for patients who have undergone interval treatment with radiation or cytotoxic chemotherapies.

c-Met gene amplification is associated with advanced stage colorectal cancer and liver metastases ☆

The c-Met proto-oncogene encodes a receptor tyrosine kinase (TK) that promotes invasive tumor growth and metastasis. Recent studies show that the presence of c-Met gene amplification is predictive for selective c-Met TK inhibitors in gastric cancer and lung cancer. In this study, we utilized a highly quantitative PCR/ligase detection reaction technique to quantify c-Met gene copy number in primary colorectal cancer (CRC) (N=247), liver metastases (N=147), and paired normal tissues. We identified no differences in c-Met gene copy number between normal colonic mucosa and liver tissue. However, mean c-Met gene copy number was significantly elevated in CRC compared with normal mucosa (P<0.001), and in liver metastases compared with normal liver (P<0.001). Furthermore, a significant increase in c-Met was seen in liver metastases compared with primary CRC (P<0.0001). c-Met gene amplification was observed in 2% (3/177) of localized cancers, 9% (6/70) of cancers with distant metastases (P<0.02), and 18% (25/147) of liver metastases (P<0.01). Among patients treated by liver resection, there was a trend toward poorer 3-year survival in association with c-Met gene amplification (P=0.07). Slight increases in c-Met copy number can be detected in localized CRCs, but gene amplification is largely restricted to Stage IV primary cancers and liver metastases. c-Met gene amplification is linked to metastatic progression, and is a viable target for a significant subset of advanced CRC.

HER kinase activation confers resistance to MET tyrosine kinase inhibition in MET oncogene-addicted gastric cancer cells

Tumor cells with genomic amplification of MET display constitutive activation of the MET tyrosine kinase, which renders them highly sensitive to MET inhibition. Several MET inhibitors have recently entered clinical trials; however, as with other molecularly targeted agents, resistance is likely to develop. Therefore, elucidating possible mechanisms of resistance is of clinical interest. We hypothesized that collateral growth factor receptor pathway activation can overcome the effects of MET inhibition in MET-amplified cancer cells by reactivating key survival pathways. Treatment of MET-amplified GTL-16 and MKN-45 gastric cancer cells with the highly selective MET inhibitor PHA-665752 abrogated MEK/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling, resulting in cyclin D1 loss and G1 arrest. PHA-665752 also inhibited baseline phosphorylation of epidermal growth factor receptor (EGFR) and HER-3, which are transactivated via MET-driven receptor cross-talk in these cells. However, MET-independent HER kinase activation using EGF (which binds to and activates EGFR) or heregulin-β1 (which binds to and activates HER-3) was able to overcome the growth-inhibitory effects of MET inhibition by restimulating MEK/MAPK and/or PI3K/AKT signaling, suggesting a possible escape mechanism. Importantly, dual inhibition of MET and HER kinase signaling using PHA-665752 in combination with the EGFR inhibitor gefitinib or in combination with inhibitors of MEK and AKT prevented the above rescue effects. Our results illustrate that highly targeted MET tyrosine kinase inhibition leaves MET oncogene-“addicted” cancer cells vulnerable to HER kinase-mediated reactivation of the MEK/MAPK and PI3K/AKT pathways, providing a rationale for combined inhibition of MET and HER kinase signaling in MET-amplified tumors that coexpress EGFR and/or HER-3. [Mol Cancer Ther 2008;7(11):3499–508]

Extracellular Metabolic Energetics Can Promote Cancer Progression

Colorectal cancer primarily metastasizes to the liver and globally kills over 600,000 people annually. By functionally screening 661 microRNAs (miRNAs) in parallel during liver colonization, we have identified miR-551a and miR-483 as robust endogenous suppressors of liver colonization and metastasis. These miRNAs convergently target creatine kinase, brain-type (CKB), which phosphorylates the metabolite creatine, to generate phosphocreatine. CKB is released into the extracellular space by metastatic cells encountering hepatic hypoxia and catalyzes production of phosphocreatine, which is imported through the SLC6A8 transporter and used to generate ATP—fueling metastatic survival. Combinatorial therapeutic viral delivery of miR-551a and miR-483-5p through single-dose adeno-associated viral (AAV) delivery significantly suppressed colon cancer metastasis, as did CKB inhibition with a small-molecule inhibitor. Importantly, human liver metastases express higher CKB and SLC6A8 levels and reduced miR-551a/miR-483 levels relative to primary tumors. We identify the extracellular space as an important compartment for malignant energetic catalysis and therapeutic targeting.

CpG Island Methylator Phenotype Associates with Low-Degree Chromosomal Abnormalities in Colorectal Cancer

Purpose: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. Experimental Design: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. Results: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). Conclusion: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.

Usefulness of carcinoembryonic antigen monitoring despite normal preoperative values in node-positive colon cancer patients

PURPOSE: The aim of our study was to determine to what extent serial carcinoembryonic antigen (CEA) monitoring is helpful in detecting colorectal cancer recurrence in patients if their preoperative serum CEA is normal. Additional major objectives of this study were to correlate CEA immunohistochemical features of the primary tumor with serum CEA levels at the time of tumor recurrence in node-positive colorectal cancer patients with low preoperative CEA values. METHODS: One hundred fourteen node-positive colorectal cancer patients with preoperative serum CEA levels of <5.0 ng/ml undergoing clinically curative operations were studied. Primary tumors were evaluated for tissue CEA using the same monoclonal antibody as used for serum CEA determinations utilizing the avidin-biotin-peroxidase immunohistochemical technique. RESULTS: The exact preoperative serum CEA value did not correlate with tumor grade, immunohistochemical CEA intensity or pattern. In the 32 patients who developed recurrent cancer, the serum CEA at recurrence was greater than 5 ng/ml in 44 percent. All such patients had CEA present in their primary tumor. There was no correlation with the exact preoperative serum CEA, the intensity of the primary tissue CEA, or the localization of such CEA and subsequent serum elevation at recurrence. CONCLUSION: Serum CEA is a useful marker in the detection of recurrent colorectal cancer despite normal preoperative values.

The Signatures of Autozygosity among Patients with Colorectal Cancer

Previous studies have shown that among populations with a high rate of consanguinity, there is a significant increase in the prevalence of cancer. Single nucleotide polymorphism (SNP) array data (Affymetrix, 50K XbaI) analysis revealed long regions of homozygosity in genomic DNAs taken from tumor and matched normal tissues of colorectal cancer (CRC) patients. The presence of these regions in the genome may indicate levels of consanguinity in the individuals family lineage. We refer to these autozygous regions as identity-by-descent (IBD) segments. In this study, we compared IBD segments in 74 mostly Caucasian CRC patients (mean age of 66 years) to two control data sets: (a) 146 Caucasian individuals (mean age of 80 years) who participated in an age-related macular degeneration (AMD) study and (b) 118 cancer-free Caucasian individuals from the Framingham Heart Study (mean age of 67 years). Our results show that the percentage of CRC patients with IBD segments (>or=4 Mb length and 50 SNPs probed) in the genome is at least twice as high as the AMD or Framingham control groups. Also, the average length of these IBD regions in the CRC patients is more than twice the length of the two control data sets. Compared with control groups, IBD segments are found to be more common among individuals of Jewish background. We believe that these IBD segments within CRC patients are likely to harbor important CRC-related genes with low-penetrance SNPs and/or mutations, and, indeed, two recently identified CRC predisposition SNPs in the 8q24 region were confirmed to be homozygous in one particular patient carrying an IBD segment covering the region.

Unusual DNA mismatch repair–deficient tumors in Lynch syndrome: a report of new cases and review of the literature

Immunohistochemical detection of DNA mismatch repair proteins and polymerase chain reaction detection of microsatellite instability have enhanced the recognition of mismatch repair-deficient neoplasms in patients with Lynch syndrome and, consequently, led to the identification of tumors that have not been included in the currently known Lynch syndrome tumor spectrum. Here, we report 4 such unusual tumors. Three of the 4, a peritoneal mesothelioma, a pancreatic acinar cell carcinoma, and a pancreatic well-differentiated neuroendocrine tumor, represented tumor types that, to the best of our knowledge, have not been previously reported in Lynch syndrome. The fourth tumor was an adrenocortical carcinoma, which has rarely been reported previously in Lynch syndrome. Three of our 4 patients carried a pathogenic germ-line mutation in a mismatch repair gene. The unusual tumor in each of the 3 patients showed loss of the mismatch repair protein corresponding to the mutation. The fourth patient did not have mutation information but had a history of colonic and endometrial carcinomas; both lacked MSH2 and MSH6 proteins. Interestingly, none of the 4 unusual tumors revealed microsatellite instability on polymerase chain reaction testing, whereas an appendiceal carcinoma from 1 of the study patients who was tested simultaneously did. The recognition of such tumors expands the repertoire of usable test samples for the workup of high-risk families. As yet, however, there are no data to support the inclusion of these tumors into general screening guidelines for detecting Lynch syndrome, nor are there data to warrant surveillance for these tumors in patients with Lynch syndrome.