Epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2/neu), members of the ErbB receptor tyrosine kinase family, are frequently overexpressed in breast cancer and are known to drive tumor growth and progression, making them promising targets for cancer therapy. Lapatinib is a selective competitive inhibitor of both the HER2 and EGFR tyrosine kinases. Although lapatinib showed significant activity in patients with HER2-positive breast cancer, the role of EGFR in the response of breast cancer to lapatinib has not been defined. Here, we examined the role of EGFR expression levels in the sensitivity of HER2-overexpressing breast cancer cells to lapatinib. Depletion of EGFR by EGFR small-interfering RNA knockdown did not affect lapatinib sensitivity in these cells, whereas treated HER2 siRNA knockdown cells became more resistant to lapatinib. We conclude that the in vitro activity of lapatinib is not dependent on EGFR expression level in HER2-overexpressing breast cancer cells.
Lapatinib is a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) with activity in HER2-amplified metastatic breast cancer (MBC). Its role in non–HER2-amplified MBC remains unclear. EGF30001, a phase III trial of lapatinib and paclitaxel versus paclitaxel and placebo, demonstrated lapatinib does not significantly benefit HER2-negative or HER2-unselected patients with MBC. Published data support interactions between steroid hormone and peptide growth factor signaling. We hypothesized that molecular subgroups may exist within EGF30001 that would benefit from lapatinib.
A blinded, retrospective biomarker evaluation was performed using immunohistochemistry to semiquantitate estrogen (ER), progesterone (PR), and EGFR expression. HER2 amplification was determined by fluorescent in situ hybridization. Effects of these biomarkers on event-free survival (EFS) were examined in patients with available tissue (n = 493).
Lapatinib improved median EFS in HER2-amplified, ER- or PR-positive MBC (n = 36; 5.7 v 4.5 months; P = .351); benefit was greater and statistically significant in HER2-amplified, ER-negative, PR-negative MBC (n = 42; 8.3 v 5.0 months; P = .007). In HER2-negative, ER-positive MBC, median EFS improvement varied by degree of PR expression (H-score): no benefit if PR-strong (n = 133; 9.3 v 7.3 months; P = .373), benefit if PR-weak (n = 50; 7.3 v 2.4 months; P = .026), and potential antagonism if PR-negative (n = 40; 3.7 v 7.2 months; P = .004). No benefit was seen in triple-negative MBC (n = 131; median EFS, 4.6 v 4.8 months; P = .255). EGFR expression was not correlated with benefit from lapatinib.
Although subgroups are small, these analyses support the hypothesis that semiquantitative determination of hormone receptor status may be a surrogate for EGFR and/or HER2 dependency.
The epidermal growth factor receptor (EGFR) is a surrogate marker for basal-like breast cancer. A recent study suggested that EGFR may be used as a target for breast cancer treatment.
A total of 706 invasive ductal carcinomas (IDC) of the breast were immunophenotyped, and 82 cases with EGFR protein expression were studied for EGFR gene amplification.
EGFR protein was expressed in 121 of 706 IDCs (17.1%); 5.9% were of luminal type, 25.3% of epidermal growth factor receptor 2 (HER-2) type, and 79.3% of basal-like tumors. EGFR gene amplification and high polysomy (fluorescent in situ hybridization [FISH]-positive) were found in 18 of 82 cases (22.0%); 41.2% of the HER-2+, EGFR+, cytokeratin 5/6- (CK5/6-) group, 11.2% of the HER-2-, EGFR+, CK5/6- group, and 19.1% of the HER-2-, EGFR+, CK5/6+ group. FISH-positive cases were detected in 8.3% of the EGFR protein 1+ expression cases, 15.9% of 2+ expression cases, and 38.5% of 3+ expression cases. In group 2, the tumors had a high Ki-67 labeling (>60%), but the patients showed better disease-free survival than those with tumors that co-expressed HER-2 or CK5/6.
EGFR-directed therapy can be considered in breast cancer patients with EGFR protein overexpression and gene amplification, and its therapeutic implication should be determined in HER-2 type breast cancer patients.
Breast neoplasms; Receptor, epidermal growth factor; Gene amplification; Protein expression
Epidermal growth factor receptor (EGFR) is involved in the development of many human malignant tumors and plays an important role in tumor growth and metastasis. Antagonists of EGFR can suppress the growth of several malignancies; however, their therapeutic effect in adenoid cystic carcinoma (ACC) is controversial.
The increased proliferation of two ACC cell lines induced by EGF-treatment was reversed by nimotuzumab. Regardless of EGF stimulation, nimotuzumab-treated ACC cells were arrested in G1 phase and showed decreased expression of Ki67. In addition, EGF activated the MAPK-dependent pathway and up-regulated the expression of matrix metalloproteinase-9 and Snail, enhancing the invasive potential of an ACC cell line (ACC-M). The effects of EGF were down-regulated by nimotuzumab treatment.
These results suggest that nimotuzumab can inhibit the growth and invasion of ACC cells induced by EGF, probably through inactivation of ERK phosphorylation. Thus, nimotuzumab should be considered as a promising novel agent for the treatment of ACC.
Epidermal growth factor receptor (EGFR); Adenoid cystic carcinoma (ACC); Nimotuzumab; Monoclonal antibody; Matrix metalloproteinase (MMP); Epithelial- mesenchymal transition (EMT); Invasion; Cancer therapy
There is strong evidence demonstrating that activation of epidermal growth factor receptors (EGFRs) leads to tumor growth, progression, invasion and metastasis. Erlotinib and gefitinib, two EGFR-targeted agents, have been shown to be relevant drugs for lung cancer treatment. Recent studies demonstrate that lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER-2 receptors, is clinically effective against HER-2-overexpressing metastatic breast cancer. In this report, we investigated the activity of lapatinib against non-small cell lung cancer (NSCLC).
We selected the lung cancer cell line A549, which harbors genomic amplification of EGFR and HER-2. Proliferation, cell cycle analysis, clonogenic assays, and signaling cascade analyses (by western blot) were performed in vitro. In vivo experiments with A549 cells xenotransplanted into nude mice treated with lapatinib (with or without radiotherapy) were also carried out.
Lapatinib dramatically reduced cell proliferation (P < 0.0001), DNA synthesis (P < 0.006), and colony formation capacity (P < 0.0001) in A549 cells in vitro. Furthermore, lapatinib induced G1 cell cycle arrest (P < 0.0001) and apoptotic cell death (P < 0.0006) and reduced cyclin A and B1 levels, which are regulators of S and G2/M cell cycle stages, respectively. Stimulation of apoptosis in lapatinib-treated A549 cells was correlated with increased cleaved PARP, active caspase-3, and proapoptotic Bak-1 levels, and reduction in the antiapoptic IAP-2 and Bcl-xL protein levels. We also demonstrate that lapatinib altered EGFR/HER-2 signaling pathways reducing p-EGFR, p-HER-2, p-ERK1/2, p-AKT, c-Myc and PCNA levels. In vivo experiments revealed that A549 tumor-bearing mice treated with lapatinib had significantly less active tumors (as assessed by PET analysis) (P < 0.04) and smaller in size than controls. In addition, tumors from lapatinib-treated mice showed a dramatic reduction in angiogenesis (P < 0.0001).
Overall, these data suggest that lapatinib may be a clinically useful agent for the treatment of lung cancer.
Lapatinib, a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR/ErbB1) and human epidermal growth factor receptor 2 (HER-2/ErbB2), is effective against HER-2–positive locally advanced or metastatic breast cancer (MBC). This phase III trial evaluated the efficacy of lapatinib in HER-2–negative and HER-2–uncharacterized MBC.
Patients and Methods
Women with MBC were randomly assigned to first-line therapy with paclitaxel 175 mg/m2 every 3 weeks plus lapatinib 1,500 mg/d or placebo. A preplanned retrospective evaluation of HER-2 status was performed using fluorescence in situ hybridization and immunohistochemistry. The primary end point was time to progression (TTP); secondary end points were objective response rate (ORR), clinical benefit rate (CBR), event-free survival (EFS), and overall survival (OS).
In the intent-to-treat population (n = 579), there were no significant differences in TTP, EFS, or OS between treatment arms, although differences in ORR and CBR were noted. In 86 HER-2–positive patients (15%), treatment with paclitaxel-lapatinib resulted in statistically significant improvements in TTP, EFS, ORR, and CBR compared with paclitaxel-placebo. No differences between treatment groups were observed for any end point in HER-2–negative patients. The most common adverse events were alopecia, rash, and diarrhea. The incidence of diarrhea and rash was significantly higher in the paclitaxel-lapatinib arm. The rate of cardiac events was low, and no difference was observed between treatment arms.
Patients with HER-2–negative or HER-2–untested MBC did not benefit from the addition of lapatinib to paclitaxel. However, first-line therapy with paclitaxel-lapatinib significantly improved clinical outcomes in HER-2–positive patients. Prospective evaluation of the efficacy and safety of this combination is ongoing in early and metastatic HER-2–positive breast cancer patients.
Lapatinib is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR/ErbB1) and human epidermal growth factor receptor 2 (HER2/ErbB2). EGFR and HER2 overexpression is associated with aggressive breast cancer with a high risk of disease relapse and death. Although lapatinib targets both EGFR and HER2, its effects on HER2 appear to be more critical. The role of lapatinib in the first-line setting remains unclear. A phase II first-line monotherapy lapatinib trial in HER2-therapy-naïve metastatic breast cancer (MBC) patients confirms efficacy in HER2-positive tumors. Retrospective analysis of a phase III, first-line MBC study confirmed incremental benefit from lapatinib and paclitaxel over paclitaxel alone in HER2-positive disease. A prospective phase III study confirms superiority of letrozole and lapatinib over letrozole alone in HER2-positive MBC. Further investigation is required to define the potential first-line role for lapatinib. Particular strengths appear to be its manageable toxicity profile, lack of cross resistance with trastuzumab, activity in central nervous system disease, and synergy in combination with other anticancer therapy. Current limitations are lack of dosing recommendations from early trials, lack of predictive biomarkers beyond HER2 status, and lack of large prospective phase III trials for HER2-positive disease in the first-line setting. The role of lapatinib in HER2-negative disease is unclear.
lapatinib; HER2; metastatic; first-line
To investigate the prognostic role of genomic gain for MET and epidermal growth factor receptor (EGFR) genes in surgically resected non–small-cell lung cancer (NSCLC).
Patients and Methods
This retrospective study included 447 NSCLC patients with available tumor tissue from primary lung tumor and survival data. EGFR and MET status was evaluated by fluorescent in situ hybridization (FISH) in tissue microarray sections.
EGFR FISH results were obtained in 376 cases. EGFR gene amplification and high polysomy (EGFR FISH+) were observed in 10.4% and 32.4% of cases, respectively. EGFR FISH-positive patients had a nonsignificant shorter survival than EGFR FISH-negative patients (P = .4). Activating EGFR mutations were detected in 9.7% of 144 stage I-II disease with no impact on survival. MET FISH analysis was performed in 435 cases. High MET gene copy number (mean ≥ 5 copies/cell) was observed in 48 cases (MET+, 11.1%), including 18 cases with true gene amplification (4.1%). MET+ status was associated with advanced stage (P = .01), with grade 3 (P = .016) and with EGFR FISH+ result (P < .0001). No patient with activating EGFR mutation resulted MET+. In the whole population, MET-positive patients had shorter survival than MET-negative patients (P = .005). Multivariable model confirmed that MET-negative patients had a significant reduction in the risk of death than MET-positive patients (hazard ratio, 0.66; P = .04).
MET increased gene copy number is an independent negative prognostic factor in surgically resected NSCLC. EGFR gene gain does not impact survival after resection.
In non-small-cell lung cancer (NSCLC), sensitivity to tyrosine kinase inhibitors (TKIs) is associated with activating mutations and genomic gain of the epidermal growth factor receptor (EGFR). Preclinical data suggested that HER3 overexpression increases sensitivity to TKIs. A total of 82 NSCLC patients treated with gefitinib (250 mg), and previously evaluated for EGFR and HER2 status by fluorescence in situ hybridisation (FISH) and DNA sequencing, and for Phospho-Akt status by immunohistochemistry, were investigated for HER3 genomic gain by FISH. Patients with high polysomy and gene amplification were considered as HER3 FISH positive (+). HER3 FISH+ pattern was significantly associated with female gender (P=0.02) and never smoking history (P=0.02). Patients with HER3+ tumours (26.8%) had a significantly longer time to progression (3.7 vs 2.7, P=0.04) than patients with HER3− tumours, but not a significantly better response rate or survival. Patients with EGFR+/HER3+ tumours had higher objective response rate (36.4 vs 9.9%, P=0.03) and time to progression (7.7 vs 2.7 months, P=0.03) than patients with EGFR− and/or HER3− tumours, but no significantly longer survival. No difference in response was observed according to HER3 status in patients with EGFR+ tumours. Patients with HER2+/HER3+ tumours had similar outcome as patients with HER2− and/or HER3− tumours. Significantly different clinical end points were not observed between patients with HER3+/P-Akt+ and HER3− and/or P-Akt− tumours. Genomic gain for HER3 is not a marker for response or resistance to TKI therapy in advanced NSCLC patients.
HER3; EGFR; tyrosine kinase inhibitor; gefitinib; non-small-cell lung cancer
To determine whether lapatinib, a dual epidermal growth factor receptor (EGFR)/HER2 kinase inhibitor, can radiosensitize EGFR+ or HER2+ breast cancer xenografts.
Methods and Materials
Mice bearing xenografts of basal-like/EGFR+ SUM149 and HER2+ SUM225 breast cancer cells were treated with lapatinib and fractionated radiotherapy and tumor growth inhibition correlated with alterations in ERK1 and AKT activation by immunohistochemistry.
Basal-like/EGFR+ SUM149 breast cancer tumors were completely resistant to treatment with lapatinib alone but highly growth impaired with lapatinib plus radiotherapy, exhibiting an enhancement ratio average of 2.75 and a fractional tumor product ratio average of 2.20 during the study period. In contrast, HER2+ SUM225 breast cancer tumors were highly responsive to treatment with lapatinib alone and yielded a relatively lower enhancement ratio average of 1.25 during the study period with lapatinib plus radiotherapy. Durable tumor control in the HER2+ SUM225 model was more effective with the combination treatment than either lapatinib or radiotherapy alone. Immunohistochemical analyses demonstrated that radiosensitization by lapatinib correlated with ERK1/2 inhibition in the EGFR+ SUM149 model and with AKT inhibition in the HER2+ SUM225 model.
Our data suggest that lapatinib combined with fractionated radiotherapy may be useful against EGFR+ and HER2+ breast cancers and that inhibition of downstream signaling to ERK1/2 and AKT correlates with sensitization in EGFR+ and HER2+ cells, respectively.
Breast cancer; epidermal growth factor receptor; HER2; radiosensitization; lapatinib
The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section.
The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays.
HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively.
We have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays.
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297
Gene-protein assay; Dual color in situ hybridization; Immunohistochemistry; HER2; Breast cancer
Salivary duct carcinomas (SDCs) and adenoid cystic carcinomas (ACCs) are the most aggressive and the most frequent carcinomas of the salivary glands, respectively. Little is known about them in terms of molecular/biochemical characterization and conventional treatments are ineffective. On cryopreserved material, we analyzed the expression/activation status of TRK-A, HER-2/neu, and KIT receptors by means of immunoprecipitation and Western blot analysis experiments, and the presence of their cognate ligands by means of Western blot analysis and/or reverse transcription-polymerase chain reaction in 9 SDCs, 12 ACCs, and 8 normal glands. The amplification status of HER-2/neu was also investigated by means of fluorescent in situ hybridization analysis on fixed material. The receptor tyrosine kinase (RTK)-deregulated profile of the SDCs was characterized by the overexpression of activated TRK-A in the presence of its ligand, and the overexpression of HER-2/neu sustained by gene amplification. The RTK signature of the ACCs was represented by the overexpression of activated KIT and TRK-A and their cognate ligands, and the overexpression of activated HER-2/neu, in the absence of gene amplification, possibly sustained by epidermal growth factor receptor heterodimerization. In conclusion, SDCs and ACCs, although sharing TRK-A autocrine loop activation, have different pathologically activated RTK-deregulated profiles that may be potential targets for pharmacological RTK inhibitors.
Aims: To investigate the importance of gene amplification and EGFR (epidermal growth factor receptor) and HER2 protein expression during the progression of adenocarcinoma of the lung.
Methods: EGFR and HER2 gene amplification was examined in atypical adenomatous hyperplasia (AAH), bronchioloalveolar carcinoma (BAC), and adenocarcinoma with mixed subtypes (MX) by chromogenic in situ hybridisation (CISH), and protein expression was examined by immunohistochemistry using paraffin wax embedded tissues.
Results: EGFR and HER2 gene amplification was found in four and two of 86 cases, respectively, and was detected only in the invasive components of MX. EGFR and HER2 protein expression was seen in 24 and 18 of 86 cases, respectively. EGFR and HER2 proteins were not expressed in AAH but were expressed in one BAC case each. EGFR and HER2 proteins were expressed in 23 and 17 of 55 adenocarcinomas with MX. EGFR and HER2 protein expression was seen more often in the invasive components than in the BAC components of MX, and increased significantly as lesions progressed from AAH to BAC, early MX, and overt MX. Because EGFR and HER2 protein expression was frequently seen without gene amplification, other mechanisms apart from gene amplification may be associated with protein expression.
Conclusions: EGFR and HER2 gene amplification may be a late event and EGFR and HER2 protein expression may be associated with the development of adenocarcinoma of the lung.
adenocarcinoma; chromogenic in situ hybridisation; epidermal growth factor receptor; HER2; lung
Glioblastoma (GBM) is the most common primary brain tumor in adults and often has amplification of the Epidermal Growth Factor Receptor (EGFR) gene. The value of EGFR as a prognostic marker in GBMs is unclear; some studies have shown an adverse correlation, while others indicated a neutral or even favorable association with longer survival. Furthermore, EGFR-amplified GBMs are usually regarded as a single subgroup of tumors, though the range of EGFR copy number varies greatly. In this study, 532 glioblastomas were analyzed for EGFR amplification via fluorescence in situ hybridization at the time of initial diagnosis. While there was no difference in survival by EGFR amplification (P = 0.33), stratification by amount of EGFR amplification showed that, surprisingly, median survival was 39% longer in the high-amplifier group (EGFR: chromosome 7 ratio > 20) versus non-amplified GBMs (P = 0.03), and 43% longer versus GBMs with low-to-moderate EGFR amplification (EGFR:chromosome 7 ratio = 2-20, P = 0.0007). Stratifying by postsurgical treatment regimens, this difference was seen only when temozolomide (TMZ) was used; tumors without amplification and high EGFR amplification both responded better to TMZ than those with low-to-moderate amplification (P = 0.01), whereas those without adjuvant therapy or adjuvant therapy without TMZ showed no survival differences (P = 0.63 and 0.91, respectively). These results suggest that glioblastomas with EGFR amplification are a heterogeneous group of tumors, and that behavior might differ according to degree of amplification, but not in a straightforward dose-response manner.
EGFR; amplification; temozolomide; glioblastoma
Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast
cancer patients to identify those most likely to benefit from herceptin-targeted
therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is
associated with reduced survival and metastasis. The most frequently used
technique for evaluating HER2 protein status as a routine procedure is
immunohistochemistry (IHC). HER2 copy number alterations have
also been evaluated by fluorescence in situ hybridization
(FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to
evaluate gene amplification is chromogenic in situ
hybridization (CISH), which has some advantages over FISH, including the
correlation between HER2 status and morphological features.
Other methodologies have also been used, such as silver-enhanced in
situ hybridization (SISH) and quantitative real-time RT-PCR, to
determine the number of HER2 gene copies and expression,
respectively. Here we will present a short and comprehensive review of the
current advances concerning HER2 evaluation in human breast
CISH; Breast cancer; HER2; Immunohistochemistry; FISH
Epidermal growth factor receptor (EGFR) gene mutation and copy number are useful predictive markers that guide the selection of non-small cell lung cancer (NSCLC) patients for EGFR-targeting therapy. This study aimed to investigate the correlation between EGFR gene mutation and copy number and clinicopathologic characteristics of Chinese patients with NSCLC. NSCLC specimens collected from 205 patients between November 2009 and January 2011 were selected to detect EGFR gene mutations with real-time polymerase chain reaction (RT-PCR) and to detect EGFR gene copy number with fluorescence in situ hybridization (FISH). EGFR mutations primarily occurred in females, non-smokers, and patients with adenocarinomas (all P < 0.001). Tissues from 128 (62%) patients were FISH-positive for EGFR, including 37 (18%) with gene amplification and 91 (44%) with high polysomy. EGFR gene mutation was correlated with FISH-positive status (R = 0.340, P < 0.001). Multivariate analysis showed that not smoking (OR = 5.910, 95% CI = 2.363–14.779, P < 0.001) and having adenocarcinoma (OR = 0.122, 95% CI = 0.026–0.581, P = 0.008) were favorable factors for EGFR gene mutation. These results show a high frequency of EGFR FISH positivity in NSCLC tissues from Chinese patients and a significant relevance between EGFR gene mutations and FISH-positive status. Among the FISH-positive samples, EGFR gene mutation occurred more frequently in samples with gene amplification compared to those with high polysomy, suggesting that EGFR mutation and gene amplification should be used as clinical decision parameters to predict response to EGFR-targeting therapy.
Epidermal growth factor receptor; gene mutation; gene copy number; non-small cell lung cancer; correlation
Epidermal growth factor receptor (EGFR) gene copy number detected by fluorescent in situ hybridization (FISH) has proven to be useful for selection of non–small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors. Here, we evaluate EGFR FISH as a predictive marker in NSCLC patients receiving the EGFR monoclonal antibody inhibitor cetuximab plus chemotherapy.
Patients and Methods
Two hundred twenty-nine chemotherapy-naive patients with advanced-stage NSCLC were enrolled onto a phase II selection trial evaluating sequential or concurrent chemotherapy (paclitaxel plus carboplatin) with cetuximab.
EGFR FISH was assessable in 76 patients with available tumor tissue and classified as positive (four or more gene copies per cell in ≥ 40% of the cells or gene amplification) in 59.2%. Response (complete response/partial response) was numerically higher in FISH-positive (45%) versus FISH-negative (26%) patients (P = .14), whereas disease control rate (complete response/partial response plus stable disease) was statistically superior (81% v 55%, respectively; P = .02). Patients with FISH-positive tumors had a median progression-free survival time of 6 months compared with 3 months for FISH-negative patients (P = .0008). Median survival time was 15 months for the FISH-positive group compared with 7 months for patients who were FISH negative. (P = .04). Furthermore, survival favored FISH-positive patients receiving concurrent therapy.
These results are the first to suggest that EGFR FISH is a predictive factor for selection of NSCLC patients for cetuximab plus chemotherapy. Prospective validation of these findings is warranted.
AIM: To analyze the pathogenetic role and potential clinical usefulness of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor 2 (HER2) in patients with advanced biliary tract cancer (BTC).
METHODS: EGFR and HER2 expression was studied in biopsy samples from 124 patients (51% women; median age 64.8 years), with advanced BTC diagnosed between 1997 and 2004. Five micrometers sections of paraffin embedded tissue were examined by standard, FDA approved immunohistochemistry. Tumors with scores of 2+ or 3+ for HER2 expression on immunochemistry were additionally tested for HER2 gene amplification by fluorescence in situ hybridisation (FISH).
RESULTS: 34/124 patients (27.4%) had gallbladder cancer, 47 (37.9%) had intrahepatic BTC and 43 (34.7%) had extrahepatic or perihilar BTC. EGFR expression was examined in a subset of 56 samples. EGFR expression was absent in 22/56 tumors (39.3%). Of the remaining samples expression was scored as 1+ in 12 (21.5%), 2+ in 13 (23.2%) and 3+ in 9 (16%), respectively. HER2 expression was as follows: score 0 73/124 (58.8%), score 1+ 27/124 (21.8%), score 2+ 21/124 (17%) and score 3+ 4/124 (3.2%). HER2 gene amplification was present in 6/124, resulting in an overall amplification rate of 5%.
CONCLUSION: Our data suggest that routine testing and therapeutic targeting of HER2 does not seem to be useful in patients with BTC, while targeting EGFR may be promising.
Cholangiocarcinoma; Gallbladder cancer; Chemotherapy; Targeted therapy
Esophageal squamous cell carcinoma (ESCC) shows a 5-year survival rate below 10%, demonstrating the urgency in improving its treatment. Alterations in epidermal growth factor receptors are closely related to malignancy transformation in a number of tumors and recent successful targeted therapies have been directed to these molecules. Therefore, in this study, we analyzed the expression of EGFR and HER2 and evaluated EGFR mutation profile as well as the presence of mutations in hotspots of KRAS and BRAF in ESCC patients.
We performed RT-qPCR, immunohistochemistry and Fluorescent in situ hybridization to determine EGFR and HER2 expression in ESCC patients, and direct sequencing and PCR-RFLP for mutations and polymorphism analysis.
Our results showed an increased EGFR mRNA expression in tumors compared to surrounding tissue (p <0.05), with 11% of the cases presenting at least a four-fold difference between tumor and paired adjacent mucosa. EGFR protein overexpression was present only in 4% of the cases. The median expression of HER2 mRNA was not different between tumors and adjacent mucosa. Still, 7% of the tumors presented at least a 25-fold higher expression of this gene when compared to its paired counterpart. Immunohistochemical analysis revealed that 21% of the tumors were positive for HER2 (scores 2+ and 3+), although only 3+ tumors presented amplification of this gene. Mutation analysis for EGFR (exons 18-21), KRAS (codons 12 and 13) and BRAF (V600E) showed no mutations in any of the hotspots of these genes in almost 100 patients analyzed. EGFR presented synonymous polymorphisms at codon 836 (C>T) in 2.1% of the patients, and at codon 787 (G>A) in 79.2% of the cases. This last polymorphism was also evaluated in 304 healthy controls, which presented a similar frequency (73.7%) in comparison with ESCC patients. The absence of mutations of EGFR, KRAS and BRAF as well as the overexpression of EGFR and HER2 in less than 10% of the patients suggest that this signaling pathway is altered in only a small proportion of patients with ESCC.
HER receptors target therapies may have the potential to be effective in only a minor fraction of patients with ESCC.
Esophageal cancer; EGFR; HER2; KRAS; BRAF; Target therapy
To assess the prognostic value of EGFR molecular characteristics of head and neck squamous cell carcinoma (HNSCC).
Patients and Methods
HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N=154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N=39) were evaluated for EGFR gene amplification by fluorescence in situ hybridization (FISH) and EGFR protein by immunohistochemical (IHC) staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (RPPA) (n=67). Tumor (n=50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR.
EGFR expression by IHC was significantly higher in the EDRN tumors with EGFR gene amplification (P<0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (p=0.019 and p=0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort (HR=2.75; 95% CI=1.26–6.00 and HR=3.29; 95% CI=1.34–8.14, respectively).
In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR.
epidermal growth factor receptor; receptor tyrosine kinase; site-specific phosphorylation; prognosis; head and neck cancer
AIM: To assess human epidermal growth factor receptor-2 (HER2)-status in gastric cancer and matched lymph node metastases by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH).
METHODS: 120 cases of primary gastric carcinomas and 45 matched lymph node metastases from patients with full clinicopathological features were mounted onto multiple-punch and single-punch tissue microarrays, respectively, and examined for HER2 overexpression and gene amplification by IHC and CISH.
RESULTS: Twenty-four tumors (20%) expressed HER2 immunohistochemically. An IHC score of ≥ 2+ was observed in 20 tumors (16.6%). HER2 amplification was detected by CISH in 19 tumors (15.8%) and in their matched lymph node metastases. A high concordance rate was found between HER2 positivity (as detected by IHC) and HER2 gene amplification (as detected by CISH), since 19 of the 20 IHC positive cases were amplified (95%). All amplified cases had 2+ or 3+ IHC results. Amplification was associated with intestinal phenotype (P < 0.05). No association with grading, staging or survival was found.
CONCLUSION: In gastric cancer, HER2 amplification is the main mechanism for HER2 protein overexpression and is preserved in lymph node metastases.
Human epidermal growth factor receptor-2; Immunohistochemistry; Chromogenic in situ hybridization; Gastric cancer
HER2 gene amplification has been detected in 10–20% of gastric adenocarcinomas. In view of the recently demonstrated clinical benefit of the anti-human epidermal growth factor receptor 2 (HER2) drug trastuzumab in the treatment of advanced gastric cancer, reliable HER2 testing is of key importance. The aim of this study was to examine HER2 status in gastro-oesophageal adenocarcinomas comparing SP3 and 4B5 immunohistochemistry (IHC) with dual probe HER2 [fluorescence in situ hybridization (FISH) and silver in situ hybridization (SISH)].
Methods and results
IHC and SISH were carried out on biopsy specimens of 146 patients with adenocarcinomas of the oesophagus and stomach. All SP3-IHC-positive cases and 91% of 4B5-IHC-positive cases were amplified. Sensitivity of SP3-IHC-positivity and 4B5-IHC-positivity for amplification was 77% and 96%, respectively. Results of FISH performed in 42 cases were identical to SISH. Amplification was heterogeneous in 73% of the adenocarcinomas; 24% of the oesophago-gastric carcinomas and 7% of distal stomach tumours were amplified.
HER2-positivity is present in a significant proportion of oesophago-gastric adenocarcinomas (24%), but at a lower rate in the distal stomach (7%). Sensitivity for amplification is higher with 4B5 IHC than with SP3. FISH and SISH yield identical results, but assessment is much easier with SISH. Our findings provide important guidance for HER2-testing in gastro-oesophageal adenocarcinomas for patients in whom anti-HER2 treatment is considered.
Anti-HER2 therapy; gastric adenocarcinoma; HER2; immunohistochemistry; in situ hybridization; oesophageal adenocarcinoma
Human epidermal growth factor receptor (HER)-2 overexpression or gene amplification is more common in high-grade or type 2 endometrial carcinomas. We assessed the discordance of HER-2 expression between primary and metastatic or recurrent endometrial carcinomas.
Materials and methods
Thirty-six primary, along with 14 metastatic and five recurrent tumors (matched to primaries), pathologically confirmed as high-grade or type 2 endometrial carcinomas, were submitted for immunohistochemistry (IHC) for HER-2. Fluorescence in situ hybridization was performed when the tumors showed HER-2 overexpression (≥2+ IHC score). The results of the IHC and fluorescence in situ hybridization assays were compared between the primary and metastatic or recurrent tumors. The relationships between HER-2 expression and clinicopathological factors or prognosis were investigated.
HER-2 overexpression and HER-2 amplification (a ratio of HER-2 copies to chromosome 17 [CEP17] copies ≥2.2) were detected in 33.3% (twelve of 36 patients) and 5.6% (two of 36 patients) of primary tumors, respectively. HER-2 overexpression was not associated with clinicopathological factors or prognosis. In 19 tumor specimens obtained from metastatic or recurrent tumors, HER-2 overexpression and HER-2 amplification were detected in 57.9% (eleven patients) and 15.8% (three patients), respectively. HER-2 overexpression tended to predict a worse prognosis.
HER-2 expression in metastatic or recurrent tumors was more frequent than in matched primary high-grade or type 2 endometrial carcinomas. Trastuzumab in combination with cytotoxic chemotherapy may represent an alternative therapeutic option for these tumors.
endometrial carcinoma; high grade; type 2; HER-2; metastatic or recurrent tumors
Responsiveness to Cetuximab alone can be mediated by an increase of Epidermal Growth factor Receptor (EGFR) Gene Copy Number (GCN). Aim of this study was to assess the role of EGFR-GCN in advanced colorectal cancer (CRC) patients receiving chemotherapy plus Cetuximab.
One hundred and one advanced CRC patients (43 untreated- and 58 pre-treated) were retrospectively studied by fluorescence in situ hybridization (FISH) to assess EGFR-GCN and by immunohistochemistry (IHC) to determine EGFR expression. Sixty-one out of 101 patients were evaluated also for k-ras status by direct sequencing. Clinical end-points were response rate (RR), progression-free survival (PFS) and overall survival (OS).
Increased EGFR-GCN was found in 60/101 (59%) tumor samples. There was no correlation between intensity of EGFR-IHC and EGFR-GCN (p = 0.43). Patients receiving chemotherapy plus Cetuximab as first line treatment had a RR of 70% (30/43) while it was 18% (10/56) in the group with previous lines of therapy (p < 0.0001). RR was observed in 29/60 (48%) of patients with increased EGFR-GCN and in 6/28 (21%) in those without (p = 0.02). At multivariate analyses, number of chemotherapy lines and increased EGFR-GCN were predictive of response; EGFR-IHC score, increased EGFR-GCN and number of chemotherapy lines were significantly associated with a significant better PFS. Response to therapy was the only prognostic predictive factor for OS. In the 60 patients analyzed for k-ras mutations, number of chemotherapy lines, increased EGFR-GCN and k-ras wild type status predicted a better PFS.
In metastatic CRC patients treated with chemotherapy plus Cetuximab number of chemotherapy lines and increased EGFR-GCN were significantly associated with a better clinical outcome, independent of k-ras status.
Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (EGFR, Her-1, or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABCB1 and ABCG2 transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1 or ABCG2 overexpressing cells and inhibited the transport of methotrexate and E217βG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [125I]Iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic.
multidrug resistance; ABCB1/P-gp; ABCG2/BCRP/MXR; EGFR tyrosine kinase inhibitor; lapatinib