Insulin-like growth factor receptor 1 (IGF1R) protein expression, mRNA expression and gene copy number in operable non-small cell lung cancer (NSCLC)

7524 Background: IGF1R is a promising target for NSCLC therapy. We have evaluated IGF1R protein expression, mRNA expression and gene copy number in primary tumors from surgically treated NSCLC patients (pts) as a reference for correlative biomarker studies in trials using IGF1R inhibitors. Methods: The study included 189 consecutive NSCLC pts who underwent curative pulmonary resection. There were 24% females, 54% squamous cell carcinomas (SCC), 29% adenocarcinomas (AC), 3% large cell carcinomas, 14% other histologies; p stage I: 41%, pII: 22%, pIII: 32% and pIV: 4%. IGF1R expression was evaluated in tissue microarrays by immunohistochemistry (IHC) with Ventana CONFIRM (N=179) and Novus (#NB600–559) anti-IGF1R Ab scored by two observers (H score 0–400). IGF1R gene copy number was assessed by FISH using customized probes (N=181). IGF1R gene expression was evaluated using qRT-PCR from 114 corresponding fresh-frozen samples. Results: Patterns of IHC staining were different for the Ventana and the Novus Ab (inverse correlation, r=-0.16, p=0.04, N=177). IGF1R protein expression detected by Ventana Ab (> median score) was more frequent in SCC (76%) than AC and other histologies (14%, p<0.001) and in pts with higher stage (p=0.03) but was not associated with survival (p=0.46). IGF1R H score by Ventana Ab, but not by Novus Ab, correlated with mRNA expression (r=0.37, p<0.001). IGF1R mRNA expression tended to be higher in SCC than in other histologies (p=0.089), but did not associate with other clinical features or survival (p=0.73). According to criteria previously established for EGFR, IGF1R gene copy number by FISH showed 5 tumors with gene amplification (2.8%), 43 tumors - high polysomy (23.8%), 87 tumors - low polysomy (48.1%), and 46 tumors - trisomy/disomy (25.4%). Pts with gene amplification/high polysomy had 3-yr survival of 60% (95% CI 47% - 74%) vs. 48% (38% - 59%) for low polysomy and 35% (21% - 49%) for trisomy/disomy pts (p=0.016). Prognostic value of IGF1R gene copy number was confirmed in the multivariate analysis. Conclusions: IGF1R protein expression is higher in SCC. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number associates with better prognosis in operable NSCLC. [Table: see text]

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Insulin-like Growth Factor Receptor 1 (IGF1R) Gene Copy Number Is Associated With Survival in Operable Non–Small-Cell Lung Cancer: A Comparison Between IGF1R Fluorescent In Situ Hybridization, Protein Expression, and mRNA Expression

Journal of Clinical Oncology ◽

10.1200/jco.2009.24.6611 ◽

2010 ◽

Vol 28 (13) ◽

pp. 2174-2180 ◽

Cited By ~ 89

Author(s):

Rafal Dziadziuszko ◽

Daniel T. Merrick ◽

Samir E. Witta ◽

Adelita D. Mendoza ◽

Barbara Szostakiewicz ◽

...

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Protein Expression ◽

Squamous Cell ◽

Copy Number ◽

Gene Copy Number ◽

Growth Factor Receptor ◽

Gene Copy ◽

Cell Versus

PurposeThe purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non–small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis.Patients and MethodsOne hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181).ResultsIGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis.ConclusionIGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.

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Gene copy number of epidermal growth factor receptor (EGFR) by fluorescent in situ hybridization (FISH) in head and neck squamous cell carcinomas (HNSCC) is closely correlated with EGFR protein levels assessed by automated quantitative protein analysis (AQUA)

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.6023 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 6023-6023

Author(s):

P. Weinberger ◽

A. Psyrri ◽

P. Kountourakis ◽

T. Rampias ◽

C. Sasaki ◽

...

Keyword(s):

In Situ Hybridization ◽

Protein Expression ◽

Protein Content ◽

Gene Amplification ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Egfr Gene ◽

Protein Levels

6023 Background: EGFR overexpression correlates with recurrence and with treatment resistance in HNSCC. The mechanisms of EGFR protein overexpression are poorly understood. Nonetheless, previous investigators have not demonstrated a correlation between EGFR gene copy number and protein content, using conventional immunohistochemistry (IHC). The aim of this study was to evaluate the relationship of EGFR gene copy number and protein expression utilizing fluorescence in situ hybridization (FISH) and AQUA, a novel, immunohistochemical method of automated quantitative in situ proteomic analysis which permits subcellular localization. Methods: A tissue microarray composed of 137 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis/Abbot) and EGFR protein expression (DAKO antibody) using AQUA analysis of EGFR staining scored on a scale of 0–255 and by conventional IHC. Agreement was assessed using kappa. Results: Sixteen (15%) of one-hundred six tumors with FISH results demonstrated EGFR high polysomy and/or gene amplification (FISH+). AQUA demonstrated a range of 3.6–102.2; protein levels assessed by AQUA in the FISH amplified cases were significantly higher (p =0.008) than in the FISH non- amplified ones. Using the EGFR 75th percentile as a cut-off, AQUA and FISH showed significant agreement (percentage of overall agreement 82%, kappa=0.458, p=0.003). To the contrary there was no concordance between FISH and conventional IHC results in this series. Conclusions: The discrepancy between EGFR gene amplification rate and protein expression by IHC reported previously may be due to the limitations and nonquantitative nature of conventional IHC. EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. No significant financial relationships to disclose.

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Mechanisms of Bcl-2 Protein Expression in Diffuse Large B-Cell Lymphoma (DLBCL).

Blood ◽

10.1182/blood.v104.11.26.26 ◽

2004 ◽

Vol 104 (11) ◽

pp. 26-26

Author(s):

Pedro Farinha ◽

Gwyn Bebb ◽

Reiner Siebert ◽

Doug Horsman ◽

Joseph M. Connors ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Expression Profiling ◽

Copy Number ◽

Gene Copy Number ◽

Copy Number Gain ◽

Gene Copy ◽

Cell Of Origin ◽

Bcl2 Protein ◽

Bcl2 Gene

Abstract Background: Bcl-2 protein expression is an important biomarker in DLBCL. Gene expression profiling also has prognostic relevance in DLBCL, establishing cell-of-origin (GCB vs non-GCB) as an important predictor of survival. Using tissue microarrays (TMA), immunohistochemical staining can be used to provide biomarker data that correlates closely with the results of gene expression profiling in predicting outcome (Hans et al, Blood 103; 275–82: 2004). The t(14;18) can be detected in DLBCL, with frequencies varying between 5–40%. The aim of this study was to clarify the different mechanisms leading to Bcl-2 expression in a cohort of patients with DLBCL. Methods: The study group consisted of 94 patients with de novo DLBCL, all with a clonal karyotype at diagnosis and treated with curative intent. A TMA was constructed with duplicate 0.6mm cores and stained for Bcl-2, CD10, Bcl-6, MUM1 and FOXP1. Cases were called positive if more than 30% of the tumor cells expressed a given protein. Cases were defined as GCB if they were CD10+. Non-GCB was defined as CD10−, MUM1+ and/or FOXP1+. Cytogenetic studies were performed routinely and locus-specific FISH was performed using commercially available Vysis probes (dual-color LSI IGH/BCL2) to detect the t(14;18). Unbalanced increases in BCL2 gene copy number were determined by comparison of BCL2 and IGH signals and correlation with the karyotype. Results: The IPI was highly predictive of overall survival (OS) (p < 0.00001). The t(14;18) was detected by both routine cytogenetics and FISH in 24 (25%) cases, but did not predict survival (p = 0.78). None of the non-GCB cases harbored a t(14;18). The t(14;18) and isolated BCL2 copy number gain were mutually exclusive. Expression of Bcl-2 protein and GCB-type immunostaining profile each predicted OS (p = 0.008 and 0.03, respectively). Expression of Bcl-2 was imperfectly correlated with either the t(14;18) or a non-GCB immunostaining profile, but was highly correlated with cases harboring an increased gene copy number for BCL2 (see Table, χ2 p=0.005). Increased BCL2 gene copy number did not predict OS (p = 0.43), a not unexpected finding as it accounts for only a proportion of Bcl-2 protein-positive cases. Cases lacking both the t(14;18) and increased BCL2 copy number were deemed cytogenetically “normal”, accounting for 47 cases. These were distributed between the GCB (42%) and non-GCB cases (62%). Of the cytogenetically “normal” cases, 35% of the GCB and 63% of the non-GCB expressed Bcl-2 protein. BCL2 Probe Result GCB(55) Non-GCB(39) n Bcl2 protein + n Bcl2 protein + t(14;18)(q32;21) 24 18 0 0 ⇑ Gene Copy# 8 6 15 14 “Normal” 23 8 24 15 Conclusions: We conclude that multiple mechanisms are responsible for Bcl-2 expression in DLBCL. Non-GCB cases do not harbor the t(14;18), more commonly have isolated BCL2 gene copy number gain and have a higher percentage of cytogenetically “normal” BCL2 protein-positive cases. The latter finding suggests a prominent role for transcriptional up-regulation of the BCL2 gene resulting from constitutive activation of NF- κB. DLBCL cases with a t(14;18) are always GCB and never show isolated BCL2 gene copy number gains, suggesting that these two events are mutually exclusive. The impact of other mechanisms (e.g. epigenetic changes, promoter hypomethylation) deregulating BCL2 expression requires further study. Bcl-2 protein expression and cell-of-origin (GCB vs non-GCB) are predictive biomarkers in DLBCL.

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Fibroblast growth factor receptor 1 (FGFR1) gene copy number gain in adenocarcinoma and squamous cell carcinoma of the lung.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.7552 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 7552-7552 ◽

Cited By ~ 1

Author(s):

Ximing Tang ◽

Yuwen Xue ◽

Xinying Su ◽

Nusrat Harun ◽

Yiran Cai ◽

...

Keyword(s):

Protein Expression ◽

Squamous Cell ◽

Copy Number ◽

Gene Copy Number ◽

Copy Number Gain ◽

Smoking History ◽

Gene Copy ◽

Primary Tumors ◽

Cell Lung Carcinoma ◽

Fgfr1 Gene

7552 Background: FGFR-1 is a novel target for therapy in non-small cell lung carcinoma (NSCLC). FGFR1 gene has been shown to be amplified in ~20% of squamous cell carcinomas (SCC) of the lung. The frequency of FGFR1 copy number gain (CNG) and gene amplification (GA) in lung adenocarcinoma (AC) is unknown, and the clinicopathological and molecular characteristics of the NSCLC tumors with FGFR1gene increase have not been fully described. Methods: We examined FGFR1 gene copy number (GCN) by fluorescent in-situ hybridization (FISH) and FGFR1 protein expression by immunohistochemistry in 475 surgically resected NSCLCs (162 SCCs, and 313 ACs) stages I-III in tissue microarrays. Three FGFR1 FISH categories were identified: a) no CNG; b) CNG, defined as ≥4 gene copies in ≥40% of cells; c) GA, defined as ratio of copies of FGFR1:CEP8 ≥2 or presence of tight gene clusters in ≥10% of cells. FGFR1 protein expression in the cytoplasm and membrane of tumor cells was quantified using H score. Results: FGFR1 GA was detected only in SCCs (14 cases, 9%). CNG was detected in both SCC (39 cases, 24%) and AC (80 cases, 26%). In AC, significantly higher frequency of CNG was detected in patients with smoking history (P=0.008) and in tumors with low differentiated histology (P=0.0005). No correlation was detected between FGFR1 CNG and mutation of KRAS and EGFR in AC. In SCC, tumors with FGFR1 CNG/GA had higher cytoplasmic FGFR1 protein expression than those with no copy changes (49±39 vs. 26±21, P<0.001), and the protein expression correlated with GCN (R=0.55; P<0.001). In multivariate analysis, patients with stage I/II SCCs having FGFR1 gene copy ≥6 or GA showed a significantly better overall survival (HR=0.26; 95% CI: 0.08-0.84, P=0.02) than patients with <6 gene copy, after adjusting for smoking, gender and tumor size. An increase on FGFR1copy number was detected in 8/45 (18%) brain metastasis compared with primary tumors. Conclusions: In NSCLC, FGFR1 GA occurs only is a small subset of SCCs (9%), whereas CNG is a relatively frequent phenomenon in both SCC (24%) and ACs (25%). FGFR1 copies ≥6 or GA is associated with better outcome in patients with surgically resected stages I/II SCCs of the lung.

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PCNA-associated factor (KIAA0101/PCLAF) overexpression and gene copy number alterations in hepatocellular carcinoma tissues

BMC Cancer ◽

10.1186/s12885-021-07994-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Anchalee Tantiwetrueangdet ◽

Ravat Panvichian ◽

Pattana Sornmayura ◽

Surasak Leelaudomlipi ◽

Jill A. Macoska

Keyword(s):

Hepatocellular Carcinoma ◽

Protein Expression ◽

Gene Amplification ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Mrna Levels ◽

Ki 67 ◽

Copy Numbers ◽

Gene Copy Numbers

Abstract Background PCNA-associated factor, the protein encoded by the KIAA0101/PCLAF gene, is a cell-cycle regulated oncoprotein that regulates DNA synthesis, maintenance of DNA methylation, and DNA-damage bypass, through the interaction with the human sliding clamp PCNA. KIAA0101/PCLAF is overexpressed in various cancers, including hepatocellular carcinoma (HCC). However, it remains unknown whether KIAA0101/PCLAF overexpression is coupled to gene amplification in HCC. Methods KIAA0101/PCLAF mRNA expression levels were assessed by quantitative real-time PCR (qRT-PCR) in 40 pairs of snap-frozen HCC and matched-non-cancerous tissues. KIAA0101/PCLAF gene copy numbers were evaluated by droplet digital PCR (ddPCR) in 36 pairs of the tissues, and protein expression was detected by immunohistochemistry (IHC) in 81 pairs of formalin-fixed paraffin-embedded (FFPE) tissues. The KIAA0101/PCLAF gene copy number alteration and RNA expression was compared by Spearman correlation. The relationships between KIAA0101 protein expression and other clinicopathological parameters, including Ki-67, p53, and HBsAg protein expression in HCC tissues, were evaluated using Chi-square test. Results Our results demonstrated that KIAA0101/PCLAF mRNA levels were significantly higher in HCC than in the matched-non-cancerous tissues (p < 0.0001). The high KIAA0101/PCLAF mRNA levels in HCC were associated with poor patient survival. The KIAA0101/PCLAF gene was not amplified in HCC, and KIAA0101/PCLAF gene copy numbers were not associated with KIAA0101/PCLAF transcript levels. KIAA0101 protein was overexpressed in the majority of HCC tissues (77.8%) but was not detectable in matched-non-cancerous tissues. Significant correlations between the expression of KIAA0101 protein in HCC tissues and p53 tumor suppressor protein (p = 0.002) and Ki-67 proliferation marker protein (p = 0.017) were found. However, KIAA0101 protein levels in HCC tissues were not correlated with patient age, tumor size, serum AFP level, or the HBsAg expression. Conclusions KIAA0101/PCLAF mRNA and protein overexpression is frequently observed in HCC but without concurrent KIAA0101/PCLAF gene amplification. Significant correlations between the expression of KIAA0101 protein and p53 and Ki-67 proteins were observed in this study. Thus, detection of KIAA0101/PCLAF mRNA/protein might be used, along with the detection of p53 and Ki-67 proteins, as potential biomarkers to select candidate patients for further studies of novel HCC treatment related to these targets.

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PCNA-associated factor (KIAA0101/PCLAF) overexpression and gene copy number alterations in hepatocellular carcinoma tissues

10.21203/rs.3.rs-39782/v2 ◽

2020 ◽

Author(s):

Anchalee Tantiwetrueangdet ◽

Ravat Panvichian ◽

Pattana Sornmayura ◽

Surasak Leelaudomlipi ◽

Jill A. Macoska

Keyword(s):

Hepatocellular Carcinoma ◽

Protein Expression ◽

Gene Amplification ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Mrna Levels ◽

Ki 67 ◽

Copy Numbers ◽

Gene Copy Numbers

Abstract Background: PCNA-associated factor, the protein encoded by the KIAA0101/PCLAF gene, is a cell-cycle regulated oncoprotein that regulates DNA synthesis, maintenance of DNA methylation, and DNA-damage bypass, through the interaction with the human sliding clamp PCNA. KIAA0101/PCLAF is overexpressed in various cancers, including hepatocellular carcinoma (HCC). However, it remains unknown whether KIAA0101/PCLAF overexpression is coupled to gene amplification in HCC.Methods: KIAA0101/PCLAF mRNA expression levels were assessed by quantitative real-time PCR in 40 pairs of snap-frozen HCC and matched-non-cancerous tissues,KIAA0101/PCLAF gene copy numbers were evaluated by droplet digital PCR (ddPCR) in 36 pairs of the tissues, and protein expression was detected by immunohistochemistry (IHC) in 81 pairs of formalin-fixed paraffin-embedded (FFPE) tissues. The KIAA0101/PCLAF gene copy number alteration and RNA expression was compared by Spearman correlation, and the relationship between KIAA0101 protein expression and other clinicopathological parameters was evaluated using Chi-square test.Results: Our results demonstrated that KIAA0101/PCLAF mRNA levels were significantly higher in HCC than in the matched-non-cancerous tissues (p<0.0001), and high KIAA0101/PCLAF mRNA levels in HCC were associated with poor patient survival. The KIAA0101/PCLAF gene was not amplified in HCC and KIAA0101/PCLAF gene copy numbers were not associated with KIAA0101/PCLAF transcript levels. KIAA0101 protein was overexpressed in the majority of HCC tissues (77.8%) but was not be detectable in matched-non-cancerous tissues. Significant correlations between the expression of KIAA0101 protein and p53 tumor suppressor protein (p=0.002), and Ki-67 proliferation marker protein (p=0.017) were found. However, KIAA0101 protein levels were not correlated with patient age, tumor size, or the expression of AFP and HBsAg.Conclusions: KIAA0101/PCLAF mRNA and protein overexpressionis frequently observed in HCC but without concurrent KIAA0101/PCLAF gene amplification. Significant correlations between the expression of KIAA0101 protein and p53 and Ki-67 proteins were observed in this study. Thus, detection of KIAA0101/PCLAF mRNA/protein might be used, along with the detection of p53 and Ki-67 proteins, as potential biomarkers to select candidate patients for further studies of novel HCC treatment related to these targets.

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Assessment of HER-2/neu, с-MYC and CCNE1 gene copy number variations and protein expression in endometrial carcinomas

Experimental Oncology ◽

10.32471/exp-oncology.2312-8852.vol-41-no-2.12973 ◽

2019 ◽

Vol 41 (2) ◽

Author(s):

L.G. Buchynska ◽

◽

O.V. Brieieva* ◽

N.P. Iurchenko ◽

◽

...

Keyword(s):

Protein Expression ◽

Copy Number ◽

Gene Copy Number ◽

Copy Number Variations ◽

Gene Copy ◽

Her 2 ◽

Endometrial Carcinomas

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MET Gene High Copy Number (Amplification/Polysomy) Identified in Melanoma for Potential Targeted Therapy

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab171 ◽

2021 ◽

Author(s):

Nisha S Ramani ◽

Ajaykumar C Morani ◽

Shengle Zhang

Keyword(s):

Targeted Therapy ◽

Gene Amplification ◽

Copy Number ◽

Kinase Inhibitors ◽

Gene Copy Number ◽

Tissue Microarrays ◽

High Copy Number ◽

Gene Copy ◽

Aberrant Expression ◽

Mesenchymal Epithelial Transition Factor

Abstract Objectives Aberrant expression of the mesenchymal epithelial transition factor (MET) gene has been observed in several malignancies, and drugs targeting the MET gene have been implicated in clinical trials with promising results. Hence, MET is a potentially targetable oncogenic driver. We explored the frequency of MET gene high copy number in melanomas and carcinomas. Methods The study group included 135 patients. Tissue microarrays were constructed with 19 melanomas and 116 carcinomas diagnosed from 2010 to 2012. We screened MET gene copy number by fluorescence in situ hybridization analysis using probes for MET gene and CEP7 as control. Results We found MET gene amplification in 2 (11%) of 19 melanoma cases, whereas 5 (26%) of 19 melanoma cases showed polysomy. For carcinomas, there was no MET gene amplification identified. However, 8 (7%) of 116 cases showed polysomy. Conclusions In our study, MET gene amplification was identified in 11% of melanomas and is relatively concordant with few reported studies. However, about 26% of the additional melanoma cases showed MET gene polysomy, which has not been reported as per our knowledge. If these results are validated with further orthogonal studies, more of the melanoma cases could potentially benefit from targeted therapy with MET tyrosine kinase inhibitors.

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