Increased Gene Copy Number Research Articles

Loss of PTEN is associated with elevated EGFR and HER2 expression and worse prognosis in salivary gland cancer

Background:Activity of the tumour-suppressor gene PTEN is reduced in different types of cancer and implicates non-responsiveness to targeted therapy. This study evaluates the gene and protein status of PTEN in salivary gland carcinomas.Methods:A total of 287 carcinomas of the major and minor salivary glands were investigated for phosphatase and tensin homologue located on chromosome 10 (PTEN) deletion and loss of PTEN expression using fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Results were correlated to clinicopathological parameters, long-term survival, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (IHC and FISH) status of the tumours.Results:Hemizygous deletions of PTEN were found in 35 out of 232 (15.1%) carcinomas, while homozygous deletions were observed in 17 out of 232 (7.3%) tumours. Phosphatase and tensin homologue located on chromosome 10 deletion was common in certain histological subtypes and especially homozygous deletion was associated with high-grade malignancy, lymph node metastases and unfavourable long-term prognosis (P<0.001). Loss of PTEN expression was present in 59 out of 273 (21.6%) carcinomas and was significantly correlated to genomic PTEN deletion, high-grade malignancy (P<0.001), increased tumour size (P=0.036), lymph node metastases (P=0.007) and worse disease-specific survival (P=0.002). Genomic PTEN deletion, in particular homogenous deletion (P<0.001) predominantly occurred in tumours with increased gene copy number of EGFR (60.0%) and/or amplification of HER2 (63.6%). Loss of PTEN expression was frequently found in tumours overexpressing EGFR (28.6%) and/or HER2 (52.6%).Conclusion:PTEN function is reduced in different types of salivary gland cancer indicating unfavourable prognosis. Its association with EGFR and HER2 signalling might affect targeted therapy.

Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

BackgroundEnzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(P)H. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH), presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S)-1-(2-chlorophenyl)ethanol from o-chloroacetophenone.ResultsAs compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3) that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR) resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C) brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by just 19% (1140 U/gCDW). The E. coli whole-cell catalyst optimized for intracellular FDH activity showed improved performance in the synthesis of (S)-1-(2-chlorophenyl)ethanol, reflected in a substantial, up to 5-fold enhancement of productivity (0.37 g/gCDW) and yield (95% based on 100 mM ketone used) as compared to the reference catalyst. For xylitol production, the benefit of enhanced FDH expression was observed on productivity only after elimination of the mass transfer resistance caused by the cell membrane.ConclusionsExpression engineering of C. boidinii FDH is an important strategy to optimize E. coli whole-cell reductase catalysts that employ intracellular formate oxidation for regeneration of NADH. Increased FDH-activity was reflected by higher reduction yields of D-xylose and o-chloroacetophenone conversions provided that mass transfer limitations were overcome.

Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5days after methanol induction compared to 6days for the wild-type. The optimum reaction temperature (60°C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50°C and pH 4). Kept at 80°C for 1h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

P5-13-07: MET and Hepatocyte Growth Factor (HGF) Increased Gene Copy Number Is Associated to Trastuzumab Failure in HER2 Positive Metastatic Breast Cancer (MBC).

Abstract Background: The ErbB2-targeting monoclonal antibody trastuzumab has remarkable efficacy in metastatic breast cancer (MBC) patients (pts) with HER2 overexpression or amplification (HER2+), either alone or in combination with chemotherapy. However, the response rate to trastuzumab is modest and not all pts derive benefit from this treatment. Predictive mechanisms of sensitivity and/or resistance are largely unknown. Recently, preclinical and limited clinical data showed that aberrant MET expression in MBC is a predictor of poor prognosis and is involved in trastuzumab resistance. Aim of the present study was to investigate whether increased gene copy number of MET or its ligand, the hepatocyte growth factor (HGF), affect trastuzumab sensitivity. Patients and Methods: This retrospective study included 130 HER2+ MBC pts treated with trastuzumab as monotherapy (N=21) or in combination with chemotherapy (N=109). Main inclusion criteria were presence of at least one measurable lesion and availability of paraffin-embedded tumor tissue from primary cancer. MET and HGF gene copy number (GCN) were assessed by fluorescence in situ hybridization (FISH). Receiver operating characteristic (ROC) analysis was used for identifying the best MET and HGF mean GCN cut-off. Results: In the whole population response rate (RR), including complete (CR) and partial response (PR) was 49.2%, disease control rate, including CR+PR+ stable disease (SD) was 76.2%, median time to progression (TTP) 9.4 months, and median survival (OS) 28.3 months. MET FISH analysis was successfully performed in all 130 cases. Median MET mean GCN was 2.96 (range 1.66−8.40), with no gene amplification. ROC curve identified a mean of 3.72 MET GCN as the optimal cut-off value for discriminating between sensitive (CR+PR+SD) and refractory pts (pts with progressive disease [PD] at the first disease assessment). MET FISH+ (N=36, mean ≥3.72) had a significantly higher PD rate (44.4% versus 16.0%; p=0.001) and a significantly shorter TTP (5.7 versus 9.9 months; HR 1.74 95% C.I. 1.16−2.62; p=0.006) than MET FISH- pts (N=94, mean &amp;lt;3.72). HGF GCN was successfully evaluated in 84 pts (64.6%). Median HGF mean GCN was 2.80 (range 1.14−6.90). ROC analysis identified a cut-off of 3.01 mean HGF GCN as the best discriminating between sensitive (CR+PR+SD) and refractory pts. HGF FISH+ (N=33, mean ≥ 3.01) had a significantly higher PD rate (30.3% versus 7.8%; p=0.007) and a non-significant shorter TTP (9.9 versus 10.5 months, HR 1.10 95% C.I. 0.70−1.74, p=0.66). Conclusions: High GCNs of MET or HGF associate with an increased risk of trastuzumab failure in HER2+ MBC. These data support a further development of combining anti-HER2 with anti-MET strategies in MBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-07.

P1-06-11: Comparison of Community and Central Her2 Assessment on Outcome of Neoadjuvant Chemotherapy in the I-SPY Trial.

Abstract Background: Her-2/neu overexpression, by immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH), is highly correlated with response to trastuzumab and these are currently the gold-standard, FDA-cleared testing methods for assigning treatment to Her-2-directed therapies. However, substantial variability has been documented between community and central laboratory IHC and FISH testing. Biologically, Her-2 overexpression may reflect increased gene copy number, gene expression and/or protein production, and these can be measured by other platforms, including comparative genomic hybridization (CGH), expression arrays and quantitative protein assays, respectively. We sought to determine the degree to which community IHC/FISH results differed from centrally-assessed IHC, FISH, and other assessment platforms within the I-SPY Trial and whether response to neoadjuvant chemotherapy (NAC) differed by platform. Methods: The I-SPY Trial enrolled 237 women 2002–06 with invasive breast tumors at least 3 cm in clinical/radiographic size who subsequently underwent anthracycline/taxane NAC, serial core biopsies and imaging. Pathologic complete response (pCR) was determined at time of surgery and 3-year follow up has been reached. Trastuzumab was given to Her2+ patients at physician discretion, based upon community IHC/FISH results, and became more widespread after 2005. Central I-SPY laboratories determined Her2 copy number by MIP array, gene expression by Affymetrix and Agilent arrays, and Her2 protein by reverse-phase protein array (RPMA). Unsupervised clustering algorithms were used to evaluate expression patterns. Composite variables were constructed for DNA, RNA and protein positivity as well as for community and central IHC/FISH. Platforms were compared and Kaplan-Meier curves were constructed to compare outcomes by platform. Results: 222 women were evaluable, though not all patients had results for all platforms. Community composite IHC/FISH was positive in 64/214 (30%) but only 41 of these (64%) were confirmed by central IHC/FISH and 4 additional cases were centrally positive despite negative community testing. Concordance was high among centrally-assessed Her2 platforms, but was lower between community IHC/protein and central RNA (90%), DNA (91%) and protein (91%). Among patients receiving trastuzumab (n=36), the pCR rate was ∼50% regardless of Her2-assessment platform; in contrast, those not receiving trastuzumab had pCR rates below 30%. Among the 64 patients deemed Her2+ by community IHC/FISH, 30 (48%) had pCR and 15 (25%) have had distant relapse. Five distant relapses have occurred despite pCR; all received trastuzumab, all were Her2 positive by multiple central platforms and 3/5 were ER-positive. Sites of distant relapse included brain, bone and viscera; only 1 of 5 had isolated brain relapse. Conclusions: Community IHC/FISH testing for Her2 expression in the I-SPY Trial overcalled Her2 positivity compared to central testing while central results were highly concordant among DNA, RNA and protein platforms. Despite the high rate of community “false positives”, relapse after pCR occurred only in central Her2 “true positives,” exclusively among those receiving trastuzumab, and was rarely isolated to CNS sanctuary sites. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-11.

P3-01-11: Increased Gene Copy Number of c-KIT and VEGFR2 at 4q12 in Primary Breast Cancer Is Related to an Aggressive Phenotype and Impaired Prognosis.

Abstract Introduction: Triple-negative breast cancer (TNBC) accounts for approximately 15% of all female breast cancer and is associated with aggressive clinical behaviour. No targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and c-KIT, have however shown some promising results for patients with TNBC. The aim of the present study was to investigate whether gains and/or amplifications of VEGFR2 and c-KIT occur in TNBC. These genes may constitute novel candidate biomarkers for selecting TNBC patients for treatment with tyrosine kinase inhibitors. Material &amp; Methods: Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and c-KIT in 83 primary human breast cancers, of which 31 were classified as TNBC. Gains were defined as ≥4 gene copies in more than 40% of the cancer cells, while amplification was defined as gene copy/CEP ratio &amp;gt;2 in more than 10% of the cancer cells. A tumour was considered FISH positive for c-KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Immunohistochemical (IHC) staining was performed for assessment of c-KIT protein expression. Results: Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were c-KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and c-KIT in nine (18%) cases for both genes. No significant difference in frequency between TNBCs and non-TNBCs was found. There was a correlation between FISH positivity for VEGFR2 and c-KIT (c2 test, P&amp;lt;0.001), and VEGFR2 and c-KIT FISH positivity correlated to ER/PgR negativity and high Nottingham histological grade (NHG). A significantly worse breast cancer specific survival (BCSS) was seen for FISH positive cases in the whole cohort as well as among untreated patients and non-TNBCs, but not among TNBC cases. Discussion: The high correlation between VEGFR2 and c-KIT FISH positivity suggests that the genes are co-amplified. Increased copy number of both genes was related to aggressive disease and a worse prognosis, and thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-11.

Increased gene copy number of KIT and VEGFR2 at 4q12 in primary breast cancer is related to an aggressive phenotype and impaired prognosis

Triple-negative breast cancer (TNBC) is associated with poor prognosis and no targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and KIT, have shown some promising results for patients with TNBC. The aim of the study was to investigate whether gains and/or amplifications of VEGFR2 and KIT, located at 4q12, occur in TNBC. Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and KIT in 83 primary human breast cancers including 31 TNBCs. Gains were defined as ≥ 4 gene copies in >40% of the cancer cells, whereas amplification was defined as CEP >2 in more than 10% of the cancer cells. A tumor was considered FISH positive for KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and KIT in nine (18%) cases for both genes, but no significant difference between TNBCs and non-TNBCs was found. FISH positivity for VEGFR2 and KIT was significantly correlated (χ(2) test, P < 0.001), and significantly related to ER negativity and high Nottingham histological grade (NHG). A significantly worse 5-year breast cancer specific survival (BCSS) was seen for FISH positive cases. Increased copy number of VEGFR2 and KIT thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category.

EGFR, HER2, survivin, and loss of pSTAT3 characterize high-grade malignancy in salivary gland cancer with impact on prognosis

Increased gene copy number (high polysomy or amplification) of EGFR and HER2 has evolved as a predictor for response to targeted therapy. STAT3 and the apoptosis inhibitor survivin represent distinct oncogenes in various human neoplasms. The purpose of this study was to evaluate protein and gene status of these biomarkers by immunohistochemistry and dual color fluorescence in situ hybridization on tissue microarrays of 286 salivary gland carcinomas in the context of clinical and histopathologic characteristics. Diverse tumor types showed overexpression and increased gene copy number of EGFR and HER2. Amplification of HER2 was found in 35.5% of salivary duct carcinomas. Protein overexpression was strongly associated with high gene copy number for both EGFR and HER2 (P < .001). Overexpression and increased gene copy number of EGFR and HER2 were correlated to high-grade malignancy (P < .001) and unfavorable prognosis (P < .001). Strong nuclear staining of survivin was found in 18.9% of tumors and was associated with high-grade malignancy (P < .001), overexpression, and high gene copy number of EGFR and HER2 (P ≤ .05) as well as unfavorable prognosis (P < .001). Overexpression of nuclear pSTAT3 was found in 28.3% of tumors and correlated with well tumor differentiation (P < .001) and favorable prognosis (P = .001). Loss or weak expression of pSTAT3 was inversely associated with overexpression of survivin (P < .001) as well as overexpression and high gene copy number of EGFR and HER2 (P < .05). Overall, overexpression and increased gene copy number of EGFR and HER2 characterize high-grade malignancy with unfavorable prognosis in salivary gland cancer. Nuclear survivin typifies aggressive tumors with worse prognosis, whereas nuclear pSTAT3 might play a role as a tumor suppressor in absence of EGFR, HER2, and survivin.

Frequent Increased Gene Copy Number and High Protein Expression of tRNA (Cytosine-5-)-Methyltransferase (NSUN2) in Human Cancers

NSUN2, also known as SAKI or MISU, is a methyltransferase which catalyses (cytosine-5-)-methylation of tRNA. The human NSUN2 gene is located on chromosome 5p15.31-33. We show that NSUN2 gene copy number is increased in oral and colorectal cancers. Protein expression levels of NSUN2 were determined by immunoblot using novel polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of the protein. In most normal tissues, NSUN2 expression levels were extremely low. On the other hand, oral and colorectal cancers typically expressed high levels of NSUN2. The level of NSUN2 was similar in interphase and mitotic cells, and immunohistochemical analysis demonstrated strong staining for NSUN2 in oral and colon cancer tissues when compared with normal tissues, providing a distinct diagnostic significance for NSUN2 in comparison with Ki-67, a widely used marker of actively proliferating cells. In addition, elevated protein expression of NSUN2 was confirmed by immunohistochemical analysis of various cancers including esophageal, stomach, liver, pancreas, uterine cervix, prostate, kidney, bladder, thyroid, and breast cancers. NSUN2 is regulated by Aurora-B, a newly developed molecular target for cancer therapy, leading us to propose that NSUN2 might become a valuable target for cancer therapy and a cancer diagnostic marker.

EGFR gene copy number increase in vulvar carcinomas is linked with poor clinical outcome

EGFR copy number increases have been frequently reported in cancer including vulvar carcinomas. Co-amplification of cancer genes plays an important role in the development of many tumour types. To better...

Clinical analysis of 11 patients with bilateral primary breast lymphoma

Objective: To investigate the clinicopathological features, treatment and prognosis of patients with bilateral primary breast lymphoma (BPBL). Methods: The clinical records of 11 patients with BPBL were collected, and the clinicopathological features, treatment and prognosis were retrospectively analyzed. Results: The medium age of 11 patients was 34 years old (ranged from 13 to 63 years). The breast tumors grew quickly, and the largest diameter of tumor was up to 20 cm. The occurrence of bilateral breast tumors was more often in synchronism than in heterochronism. The incidence rates of B symptoms and high level of serum LDH were both low in BPBL patients, and the axillary lymph node metastasis and distant metastasis were frequent. The BPBL was originated mainly from B cells, and the most common pathological grade of BPBL was grade high/intermediate. The median survival of BPBL was 33 months. Furthermore, chemotherapy played an important role in the treatment of BPBL. Conclusion: In consideration of BPBL which has a high rate of diagnostic errors, the clinicopathological features and medical imagings should be applied in the diagnosis of BPBL. The major modality used in the treatment of BPBL is chemotherapy. DOI:10.3781/j.issn.1000-7431.2011.10.013

Abstract B177: The use of Oncotest molecular database to identify amplified and overexpressed drug target genes.

Abstract Background: Carcinogenesis is characterized by complex genomic rearrangements including gene copy number variations, gene fusions or somatic mutations and may contribute to tumor progression. Increased gene copy number is one of the mechanisms shown to cause over-expression and over-activation of genes and signaling pathways involved in tumor progression. Such alterations offer a rationale to develop drugs specifically blocking these over-expressed genes. Recently, we characterized more than 250 patient-derived tumor xenografts in nude mice on DNA, RNA and protein levels using high throughput technologies. In the present study, we used this database to identify putative target genes that are amplified and over-expressed in our tumor models. Method: A total of 176 patient-derived tumor models representing 26 tumor types have been investigated. DNA was analyzed using Affymetrix genome wide SNP6.0 arrays and RNA transcripts by Affymetrix HGU-133 plus 2.0 arrays. Analysis was done using R-based statistical tools and Pathway Studio. Gene variations were considered relevant when having an increased gene copy number equal to 4 and log of signal intensity from microarray higher than 12. Result: The DNA analysis of the 176 tumor models allowed identification of a total of 2003 different genomic regions with a copy number gain equal to 4. The median number of genomic regions with copy number gains is 15 per tumor model but was shown to greatly vary between tumor types (colon tumors having the lowest median number ∼ 5). The genomic region gains were observed frequently for chromosomes 3, 5, 8, 17 and 18. The size of the 2003 regions with increase copy number gain varied from 100kb to 89mb and included a total of 16.558 protein coding sequences coding. Transcript analysis using Affymetrix data revealed that among these 16.558 coding sequences, 1609 (10%) have a log2 intensity signal above 12 and were considered over-expressed. The pathway studio analysis revealed genes coding for proteins with trans-membrane domain, receptors, kinases and oncogenes. As expected, we identified tumor models with amplified regions containing overexpressed growth factors or receptors such as AREG, EGFR, HER2, c-Met and FGFR1. The analysis also showed new amplified and over-expressed genes associated with cellular growth. These may be putative drug targets. Conclusion: In the present study, we showed that our patient-derived tumor xenograft panel contains representative tumor models with well know amplified and over-expressed genes. The analysis also revealed tumor models with new drug targets genes. The strategies of blocking them at RNA or protein level in appropriate tumor models could contribute to identification of new drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B177.

Increased gene copy number of ERG on chromosome 21 but not TMPRSS2–ERG fusion predicts outcome in prostatic adenocarcinomas

The role of TMPRSS2–ERG gene fusion in prostate cancer prognostication remains controversial. We evaluated the prognostic role of TMPRSS2–ERG fusion using fluorescence in situ hybridization analysis in a case–control study nested in The Johns Hopkins retropubic radical prostatectomy cohort. In all, 10 tissue microarrays containing paired tumors and normal tissues obtained from 172 cases (recurrence) and 172 controls (non-recurrence) matched on pathological grade, stage, race/ethnicity, and age at the time of surgery were analyzed. All radical prostatectomies were performed at our institution between 1993 and 2004. Recurrence was defined as biochemical recurrence, development of clinical evidence of metastasis, or death from prostate carcinoma. Each tissue microarray spot was scored for the presence of TMPRSS2–ERG gene fusion and for ERG gene copy number gains. The odds ratio of recurrence and 95% confidence intervals were estimated from conditional logistic regression. Although the percentage of cases with fusion was slightly lower in cases than in controls (50 vs 57%), the difference was not statistically significant (P=0.20). The presence of fusion due to either deletion or split event was not associated with recurrence. Similarly, the presence of duplicated ERG deletion, duplicated ERG split, or ERG gene copy number gain with a single ERG fusion was not associated with recurrence. ERG gene polysomy without fusion was significantly associated with recurrence (odds ratio 2.0, 95% confidence interval 1.17–3.42). In summary, TMPRSS2–ERG fusion was not prognostic for recurrence after retropubic radical prostatectomy for clinically localized prostate cancer, although men with ERG gene copy number gain without fusion were twice more likely to recur.

MET Signaling Pathway: A Rational Target for Cancer Therapy

In Journal of Clinical Oncology (JCO), two groups of investigators provide important evidence for the critical role of MET in malignant transformation. Graziano et al report a dose-proportional risk of recurrence and decreased survival associated with increasing MET gene copy number in stages II to III gastric adenocarcinoma. Lennerz et al show that MET amplification in gastroesophogeal carcinomas is uncommon, observed in only 2% of their patient cohort. However, an increase in MET gene copy number was associated with higher presenting tumor stage and grade as well as shorter median survival. These two reports add to a growing body of evidence that MET is a key driver of oncogenic transformation in a defined subset of cancers. This Understanding the Pathway article will provide an overview of the MET signaling pathway, discuss the role of MET in malignancy, and consider the potential for targeting MET in cancer therapy. MET is an integral plasma membrane protein that relays signals from the extracellular environment into the cytoplasm (Fig 1). MET is activated when its extracellular domain binds to hepatocyte growth factor (HGF), also known as scatter factor. This protein is primarily expressed on epithelial cells, whereas HGF is secreted by mesenchymal cells and bound in an inactive form to heparin proteoglycans within the extracellular matrix. The HGF polypeptide is inactive in its initial form and must be cleaved into a disulfide-linked heterodimer by an extracellular protease—HGF activator—to acquire MET-binding activity. HGF activator is a serine protease that is produced in the liver but localized and activated selectively at sites of tissue injury and in cancer. The mesenchymal-epithelial communication mediated by HGF-MET signaling is critical to the formation of threedimensional body structures (morphognesis) through cellular behaviors collectively described as invasive growth: proliferation and survival (resistance to apoptotic signals), increased cell motility, cell dissociation (scattering), epithelial tubulogenesis, infiltration of tissues, and stimulation of angiogenesis. These MET-dependent behaviors are essential for normal processes such as embryogenesis, organ regeneration, and wound healing. Cancers with increased MET activity commandeer this machinery to facilitate tumor growth and invasion/metastasis. The cascade of molecular events triggered by HGF begins with MET kinase activation within the receptor’s cytoplasmic tail. Two tyrosine residues within the kinase domain undergo transautophosphorylation to create a fully activated enzyme. Two additional tyrosine residues are subsequently phosphorylated, resulting in a high-affinity binding motif that recruits a range of Src homology 2 domain– containing proteins, which include nonenzymatic adaptor proteins such as growth factor receptor– bound protein 2 (GRB2) and GRB2-associated binding protein 1. GRB2-associated binding protein 1 is phosphorylated by MET and cooperates with GRB2 and other adaptors to serve as a scaffold for additional signaling proteins, including phosphoinositide 3-kinase, phospholipaseC1, STAT3, focal adhesion kinase (involved in motility), and the tyrosine MET

Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania.

Leishmania parasites cause a spectrum of clinical pathology in humans ranging from disfiguring cutaneous lesions to fatal visceral leishmaniasis. We have generated a reference genome for Leishmania mexicana and refined the reference genomes for Leishmania major, Leishmania infantum, and Leishmania braziliensis. This has allowed the identification of a remarkably low number of genes or paralog groups (2, 14, 19, and 67, respectively) unique to one species. These were found to be conserved in additional isolates of the same species. We have predicted allelic variation and find that in these isolates, L. major and L. infantum have a surprisingly low number of predicted heterozygous SNPs compared with L. braziliensis and L. mexicana. We used short read coverage to infer ploidy and gene copy numbers, identifying large copy number variations between species, with 200 tandem gene arrays in L. major and 132 in L. mexicana. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in L. infantum, four in L. mexicana, two in L. braziliensis, and one in L. major. A significant bias against gene arrays on supernumerary chromosomes was shown to exist, indicating that duplication events occur more frequently on disomic chromosomes. Taken together, our data demonstrate that there is little variation in unique gene content across Leishmania species, but large-scale genetic heterogeneity can result through gene amplification on disomic chromosomes and variation in chromosome number. Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism.

Molecular Characterization ofRht-1Dwarfing Genes in Hexaploid Wheat

The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer "GA-insensitive" dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.

Abnormality of the hepatocyte growth factor/MET pathway in pulmonary adenocarcinogenesis

Background Signaling mediated by hepatocyte growth factor (HGF)/MET promotes multiple biological activities, including cell proliferation, motility, invasion, angiogenesis, and morphogenesis. Overexpression of HGF and MET and an increase of the MET gene copy number have recently been found in various cancers that had a poor outcome. Here we investigated the copy number of the MET gene and expression of MET and HGF in small pulmonary adenocarcinomas. Methods Tumor tissues were obtained from 106 pulmonary small adenocarcinomas 2 cm or less in diameter. MET gene copy number, and the expression of MET and HGF, were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry, respectively. Results MET FISH-positive signals were observed in 11 (10.4%) of 106 cases. One case (0.9%) showed gene amplification and 10 (9.4%) exhibited high polysomy. High immunoreactivity for MET and HGF in tumor cells was found in 30 (28.3%) and 19 cases (17.9%), respectively. HGF was also expressed in stromal cells in 32 cases (30.2%). No cases of non-invasive adenocarcinoma (adenocarcinoma in situ, localized bronchioloalveolar carcinoma) showed MET FISH-positive signals or high expression of HGF in the tumor cells. Expression of both MET and stromal HGF was stronger in invasive than in non-invasive adenocarcinoma. MET FISH-positive signals and high immunoreactivity for MET and HGF in tumor cells were associated with factors indicative of poor prognosis such as pleural invasion, vascular invasion, lymphatic permeation, lymph node metastasis, and nuclear grading. Univariate and multivariate analyses that included these factors showed that all statuses except for MET and HGF immunoreactivity were significantly associated with an increased risk of death. However, multivariate analysis revealed no independent factors related to poor prognosis. Conclusion Our results suggest that abnormality of the HGF/MET pathway occurs during the course of progression from non-invasive to invasive pulmonary adenocarcinoma. An increased MET gene copy number is indicative of a poor outcome in patients with small pulmonary adenocarcinomas.

Gene amplification and protein overexpression of EGFR and ERBB2 in sinonasal squamous cell carcinoma

Sinonasal squamous cell carcinomas (SNSCCs) are rare tumors with no etiologic link to tobacco or alcohol, as opposed to other squamous cell carcinomas of the head and neck. Despite improvements in the field of surgery and radiotherapy, patients with these tumors still face a very unfavorable prognosis, partly because of their localization in a complex anatomic area, which has special relevance for surgery and postoperative treatment. Therefore, there is a need for new therapeutic possibilities for patients with these tumors. Gene copy numbers of epidermal growth factor receptor (EGFR) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2) were analyzed by fluorescence in situ hybridization and multiplex ligand-dependent probe amplification, and protein expression was evaluated by immunohistochemistry in 54 SNSCC specimens. The results were correlated with clinicopathologic and follow-up data. EGFR gene copy number increases were observed in 20 of 45 tumors (44%), and 21 of 54 tumors (39%) had EGFR protein overexpression. Eight of 38 tumors (21%) had ERBB2 copy number increases, and 4 of 54 tumors (7%) exhibited elevated protein expression levels. Both copy number increases and protein overexpression of EGFR and ERBB2 were mutually exclusive. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were absent in 37 tumors that were analyzed. A substantial proportion of SNSCCs carried alterations in EGFR or ERBB2. Together with the absence of KRAS mutations, these findings indicate that therapies targeting these molecules may be promising additions to the therapeutic options for patients with SNSCC.

Relationship between epidermal growth factor receptor gene copy number and protein expression in oral cavity squamous cell carcinoma

This study was designed to explore the relationship between epidermal growth factor receptor (EGFR) copy number and EGFR protein expression in oral cavity squamous cell carcinoma (OSCCs) in Taiwan. A total of 160 oral cavity squamous cell carcinomas were examined for EGFR protein overexpression using immunohistochemistry and for copy number using a fluorescence in situ hybridization (FISH) assay. Overexpression and increased gene copy numbers of EGFR were found in 75 (46.88%) and 50 (31.25%) cases, respectively. The concordance rate for EGFR gene amplification and protein overexpression was 100%. EGFR overexpression was associated with a poor prognosis both in terms of disease-free survival (DFS) and overall survival (OS). On the other hand, the association between an increase in EGFR gene copies and DFS or OS was insignificant. This was despite the observed significant associations between gene copy number and tumor stage, depth of tumor invasion, lymph node metastasis, bone invasion and perineural invasion. EGFR protein overexpression is closely related to EGFR copy number. Standard methodological and interpretation criteria need to be established that allows EGFR copy number combined with EGFR protein expression to be determined in a manner that allows individualized EGFR targeted therapy in OSCC patients.

Mechanisms of Acquired Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors and New Therapeutic Perspectives in Non Small Cell Lung Cancer

EGFR somatic mutations define a subset of NSCLCs that are most likely to benefit from EGFR tyrosine kinase inhibitors (TKIs). These tumors are dependent on EGFR-signaling for survival. Recently, tyrosine kinase domain somatic mutations have been approved as criterion to decide first-line therapy in this group of advanced NSCLCs. Anyway, all patients ultimately develop resistance to these drugs. Acquired resistance is linked to a secondary EGFR mutation in about a half of patients. Uncontrolled activation of MET, another tyrosine kinase receptor, has been implicated in neoplastic invasive growth. MET is overexpressed, activated and sometimes mutated in NSCLC cell lines and tumor tissues. MET increased gene copy number has also been documented in NSCLC and has been studied as negative prognostic factor. It has also been found in about 20% of patients developing acquired resistance to TKIs inhibitors. In this group, it seems to display a new mechanism, which is able to mark tumor independence from EGFR signaling. The study of delayed resistance mechanisms could lead to the development of new therapeutic strategies. Different molecular alterations could be specifically targeted in order to extend disease control in this group of NSCLCs with distinct clinical and molecular features. EGFR irreversible inhibitors, MET inhibitors and dual EGFR/VEGFR inhibitors represent one of the most challenging issues in current clinical research. Ongoing clinical trials and future perspectives are discussed.