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EGRF inhibitors and effect in lung cancer: scientific background Great article on recent developments EGRF inhibitors (Epidermal Growth Factor Receptor) in the treatment of lung cancer. We translated this English article by Professor Giuseppe Giaccone (VU Amsterdam). Update January 10, 2011

January 10, 2011: we have this article from Prof. Giaccone 2005 once again highlighted although there might be a different and changed. But the effect of EGRF inhibitors will remain the same, we assume. We pick this item up because recently also in lung cancer provided that certain receptors on the tumors are necessary for effective use of Tarceva and Iressa for example. But see this article by Dr. Giaccone.

December 23, 2005: David translated this great scientific article on EGRF inhibitors for us, for which many thanks. The author Professor Giuseppe Giaccone has been working in the VU - Amsterdam (020-4444444). The co-author Jose Antonio Rodriguez is also active in the VU. We have this product, its translation, is presented to a medical expert but it may still be errors removed. We also Prof. Giaccone permission but no reply received. But because the article interesting and oi belangijrk ljikt and openljik is gepulbiceerd place this article here we do but only for OPS members. But please do this with care and always do it in consultation with your doctor if you have something to do with this information.

It is noteworthy in this article that the effect of EGRF inhibitors in lung Asian patients is significantly higher than in lung cancer patients living in a Western society. In Asia, profit up 25% EGRF inhibitors as Iressa and Tarceva, in the west only 10%. Giaccone attributes this to a certain gene structure that would have prevented Asians and Westerners do not. We have some English medical terms or statements that we could not be translated, but for whom certain words have a clearer translation we feel is recommended.

EGFR inhibitors: what have we learned from the treatment of Lung Cancer?
Giuseppe Giaccone, Jose Antonio Rodriguez

Summary
Tyrosine Kinase Inhibitors that work against the Epidermal Growth Factor Receptor (EGFR) are the first molecules oriented funds in the U.S. and other countries should be approved for the treatment of advanced non-small-cell lung cancer after failure of chemotherapy. Some patient characteristics such as: never smoked, female, East Asian origin, adenocarcinoma histology, and broncho-alveolar subtype, are associated with a greater benefit from treatment with EGFR inhibitors.
Recent research has identified genetic mutations that affect the kinase domain of EGFR, which are related to the response to inhibitors.

Most EGFR mutations predict a greater benefit of treatment in comparison with "wild-type receptors and are correlated with clinical features associated with a better result, some EGFR mutations lead to resistance. The analysis of material usually available in lung cancer patients, using techniques such as direct sequencing to determine EGFR mutation status, can be technically challenging. In this context, high numbers of EGFR copies, and EGFR proteins are detected by immunohistochemistry, also be used to select patients who could benefit from treatment. This requires prospective validation of biological sensitivity and clinical markers be performed

Introduction
Lung cancer is the leading cause of cancer related deaths in the Western world and the mortality rate is rising rapidly in Asia in 2002 was 1.2 million worldwide cancer deaths due to lung cancer [1].
First are two main types of lung cancer: non-small cell lung cancer (NSCLC) and small cell lung cancer. More than 50% of NSCLC patients are candidates for systemic chemotherapy, either because of advanced disease or as adjuvant or neoadjuvant therapy to local therapy. Chemotherapy works but moderately in NSCLC, and in the last few years, several drugs aimed at specific targets within the cancer cell to show efficacy in NSCLC. [2 3]

There are two molecules targeted agents approved for the treatment of advanced NSCLC: gefitinib (Iressa ®, AstraZeneca, Wilmington, DE) and erlotinib (Tarceva ®, OSI Pharmaceuticals Inc., Melville, NY). Both drugs are small molecules belonging to the Class of quinazolinaminen and the tyrosine kinase of the epidermal growth factor receptor (EGFR) by inhibiting competing with ATP for the ATP-binding site [4].
Besides these two relatively selective tyrosine kinase inhibitors (TKIs) of EGFR, in advanced NSCLC are also other TKIs tested with a broader spectrum of activity, and monoclonal antibodies against the extracellular domain of the receptor are those tested. [4]

Among the broader spectrum TKIs include lapatinib and canertinib, acting on several members of the ErbB family of receptors, and ZD6474 and AEE788, in addition to EGFR, the vascular-endothelial growth factor receptor (VEGFR) prevent. After failure of chemotherapy to erlotinib and gefitinib in about10% of white western patients with NSCLC tumors cause a major response, and in approximately 25-30% of Japanese patients (gefitinib). The response rates of the monoclonal antibody against EGFR, cetuximab (Erbitux ®, ImClone Systems / Bristol-Myers Squibb) , seems to indicate similar results, but these are not yet available experience with Asian patients. [4)

Biological Aspects of EGFR mutations associated with tyrosine kinase inhibitor responsiveness.

A major breakthrough in the field of EGFR-targeted therapy started in 2004 with the identification of somatic changes in the EGFR gene, which is highly correlated with a favorable clinical response of NSCLC patients to treatment with gefitinib and erlotinib. [5.6, 7] These genetic changes consisted SMALL CHANGES IN THE FRAME OF POINT MUTATIONS IN EGFR-exon 18-24, which the kinase domain of the protein coding and grouped into two mutational hot spots in the EGFR gene. The most frequently detected changes were small deletions in exon 19 that the amino acids 747-750 (Leu-Arg-Glu-Ala) elimination, which are located around the active site of the kinase, and point changes in exon 21 leading to the amino acid substitution Leu858 to Arg, a residue located in the activation circuit. [5,6,7]

The in vitro analysis of the phenotypic effects associated with the expression of mutant EGFR proteins provided data on the molecular and cellular mechanisms underlying the increased responsiveness to gefitinib and erlotinib in NSCLC patients with tumors that have EGFR mutations. The efficiency of these drugs was significantly higher in terms of blocking the activity of the receptor and reducing the viability of the cell, that cell lines that had mutant EGFR gene. Transfection experiments in cell lines derived from NSCLC [5,8,9] showed that although mutant EGFR more pronounced and persistent activation of ligand binding to wild-type receptors undergo their efficient EGFR activity is inhibited by gefitinib and erlotinib [6.7 , 8]. The molecular basis for this differential sensitivity of mutant EGFR proteins remains to be determined, since wild-type and mutant receptors do not appear to differ significantly in their ability to use these inhibitors in vitro to interact. [10] Important enough, the binding of ligand to the mutant EGFR lead to selective activation of downstream signaling pathways that promote cell survival, such as protein kinase B (Akt) and signal transducers and activators of transcription (STAT) pathways. [8.9]

The pro-survival signals generated by these downstream signaling molecules are transferred seem to be essential for NSCLC cells with mutant EGFR expression, as these cells massive cell death underwent after pharmacological inhibition of Akt and STAT signaling or RNA interference-mediated specific downregulation of the mutant receptor [8] These observations suggest that the increased efficacy of gefitinib and erlotinib in NSCLC patients with EGFR mutations at least in part engineered by the degradation of the pro-survival signals that tumor cells with a mutant receptor have become dependent. [8 , 1911]

The EGFR Mutation Status as a Prognostic Marker of Tyrosine Kinase Inhibitor Response.

The first studies that evaluated EGFR mutations, selected patients with severe and prolonged response. [5.6]. Mutations were most frequently detected in a subpopulation of NSCLC patients with characteristics which went with a more consistent result: women, non smokers, patients of Japanese descent, and patients with adenocarcinoma histology and especially bronchio-oalveolair carcinoma. [4, 12]. The largest currently available database of patients treated with a single drug, comes from the BR.21 study, a randomized trial in which erlotinib was compared with best palliative care in patients with advanced NSCLC with chemotherapy in first or second line failed had. This study showed that erlotinib significantly improved survival, although the response was only 8.8% [13].

This modest response would not only be attributed to such a significant survival benefit. It should be noted that a large proportion of patients in this study or whether there was a lack of response or stable disease, whereas one of the major effects of EGFR inhibitors in preclinical studies, the capacity was observed in cell proliferation reduction [14], this would indeed be an importanteffect can be. In NSCLC cell lines, apoptosis is only observed when both the extracellular signal-regulated kinase and the Akt pathways by EGFR inhibition were blocked [14, 15] while in most cell lines was not possible and only inhibition of proliferation was observed. Of the 731 patients in 197 study BR.21 was determined using the EGFRstatus Immunohistological examination (IHC), fluorescence in situ hybridization (FISH) and mutation analysis. In multivariate analysis, the intended response depended significantly associated with adenocarcinoma histology, never-smoking status, and EGFR expression by IHC. [13].

Univariate analysis showed that survival was significantly longer in the erlotinib group when EGFR expression was high and the tumor cells a large number of copies of EGFR genes had been detected through FISH. However, the influence of EGFR status by any of the three methods used determined, multivariate analysis showed no significant. This study caused some doubt as to the validity of the mutation status of EGFR as a predictor of response and survival in larger patient populations. In this study, however, many not previously reported mutations identified (24/45, 53%). Hence these data may need to be confirmed, perhaps by a cross-validation analysis. The functional implications of the new mutations should be confirmed.

The number of patients who had anti-EGFR therapy benefit in this study [13] and other large clinical trials (16,17] exceeded the number of NSCLC patients with one based on the prevalence of EGFR mutations that were found in previous studies, expected that they possessed a mutant EGFRgen. Approximately 40-50% of patients in two large phase II trials of gefitinib showed clinical improvement, which is equivalent to the number of patients with good response plus stable disease [16.17]. This observations indicated that, although the presence of an EGFR mutation is a favorable response to treatment with gefitinib and erlotinib can reliably predict a subgroup of patients taking these drugs would benefit, not selected would be based on EGFR mutation status. The close relationship between the presence of mutant EGFR gene and clinical response to TKIs is consistently confirmed (Table 1). However, as the number of NSCLC patients receiving EGFR mutation analysis have increased, it clearer that a small but visible subset of patients with mutations not gives response to TKI treatment.

Conversely, it is also clear that some patients who respond well to treatment with gefitinib and erlotinib have wild-type EGFR. These observations may indicate that the procedures to evaluate the EGFR mutation status should be optimized, and that additional markers should be established to clearly identify each of the NSCLC patients to identify those of anti-EGFR therapy can benefit. Similar to findings in patients with gastrointestinal stromal tumors that mutations in the C-KIT gene showed, it seems that EGFR mutations in NSCLC different sensitivities to TKIs can evoke.
In two reports from Asia (Taiwan and Japan) where EGFR mutations occur more frequently than in the Western world seem to change in exon 19 of EGFR gene to provide more sensitivity than point changes in exons 20 and 21. [18.19] Data the relative rarity of EGFR mutations in white Westerners, it is currently difficult to discuss these findings to non-Asian patients to translate.

The Optimization of EGFR Mutation Analysis
It has been argued that the presence of non-detected EGFR mutations in some of the beneficial responses to TKIs may explain those observed in NSCLC patients who carry apparently wild-type EGFR [12.20]. Although several of the NSCLC series on the presence of EGFR mutations are analyzed either wholly or partly of frozen tissue samples (especially surgical patients), the most common type of tissue for analysis was available, DNA from formalin-fixed, paraffin embedded tissues is achieved. Following the fixation procedure, these are usually degraded DNA samples of poor quality, sometimes difficult to analyze. On the other hand, in patients with advanced lung cancer is often only a few diagnostic tools are available, and it is often cytology which makes it difficult to direct sequencingto apply because of an insufficient number of tumor cells in the specimen. In addition, wild-type EGFR DNA sequences of normal cells in the sample for the presence of mutations can mask. In this context there is some evidence that the most common method of polymerase chain reaction (PCR) amplification followed by direct sequencing, mutations could not possibly discover who can be identified using a different method of analysis, such as single-strand Conformation Polymorphism [21].

Furthermore, the analysis can be improved by using new techniques based on denaturing high performance liquid chromatography, which for the detection of EGFR mutations seem to be more sensitive than direct sequencing [22], and by developing methods to specifically most common types of mutations to determine, based on simple PCR and restriction of enzymvertering.Voorts new data indicate that the use of laser capture microdissection to select for tumor cells could lead to the identification of EGFR mutations that are undetectable if not microscopic removed tumor tissue is analyzed [23]. Most of these additional procedures are technically demanding and can be difficult to implement in many clinical laboratory settings. In summary, the possibility that some EGFR mutations difficult to detect with the use of easy to implement, technically straightforward approach stresses the importance of establishing additional prognostic markers, especially those not dependent on PCR-based analysis of DNA from tumor tissue is removed.

Additional Markers for Response to EGFR Tyrosine Kinase Inhibitors in NSCLC to predict.

In other molecules targeted forms of therapy, such as treatment of metastatic breast cancer with the anti-human epidermal growth factor receptor (anti-HER2) antibody trastuzumab (Herceptin ®, Genentech Inc.., San Francisco, CA) , is overexpression of the target protein ( ie HER2), a critical marker that is used to select patients who will benefit from treatment with EGFR TKIs. [24]. In contrast, initial studies being NSCLC patients that the treatment outcome of gefitinib in patients with both HER2 and EGFR overexpression showed, was not different compared with patients who had no HER2 overexpression. [25.26). Similar results were obtained in the first phase II study of erlotinib (27). In a recent report (28) with the EGFR status determined by three different methods: direct sequencing to examine the presence of mutations, FISH for the number of copies of the EGFR gene to establish and IHC the levels of EGFR protein expression evaluation. In this study, "EGFR Positivity" in any analysis of these significantly associated with a better response to gefitinib, but a better survival was only found in a large number of copies of the EGFR gene in multivariate analysis.

These results are generally consistent with the results obtained in patients treated with erlotinib in the BR.21 study. [29) Consistent results that the predictive value of determining the copies of the EGFR gene support have been obtained in independent studies or FISH (30) or a quantitative PCR determinations. [23]. Remarkably, a significant correlation between the presence of EGFR mutations and a high number of EGFR copies [23, 28, 31], suggesting that mutant allele of the EGFR gene is selectively increased in tumors, such as mutant EGFR NSCLC cell lines seen. [9.23]. Although potentially interesting, was the increased number of copies of the gene based on a scoring system of six categories by using FISH analysis and an arbitrary cut-off value by opposite transcription PCR. This analysis will require confirmation in prospective studies and confirmation by other laboratories.

Members of the ErbB family of receptors (other than EGFR) that might "heterodimerize" (?) EGFR, such as HER2, were also tested as potential predictive markers for anti-EGFR therapy. In the same way, signaling molecules downstream of the EGFRroute function, such as K-ras and Akt, thought to function as a prognostic markers .. Although the response to gefitinib seems to be independent of the level of HER2, as determined by IHC [25], is still in NSCLC patients recently reported the presence ofsomatic changes the protein structure of the HER2 kinase domain changes. [32.33). These mutations are small insertions and duplications that focus on a field similar to HER2 exon 19 deletions in the EGFR gene. HER2 mutations seem to be more frequent in patients with clinical features similar to the subpopulation of patients with EGFR mutations, it is remarkable that they are never the same EGFR mutations occur. The prevalence and functional relevance of HER2 mutations should be further explored for their value as markers for anti-EGFR therapy to be determined.

Interestingly, mutations in EGFR or HER2 genes seem mutually exclusive to work with K-ras mutations. [34,35,36,37]. Furthermore, the presence of K-ras mutations associated with a lack of response to treatment with gefitinib erlotinib.[35] and therefore, the presence of a K-ras gene mutation, occurring in approximately 30% of NSCLC patients ontdekt[38] is probably a useful marker for those patients to select from an anti-EGFR therapy will benefit. In a large, randomized study of chemotherapy with or without erlotinib as first line therapy for patients with advanced NSCLC were [39] EGFR mutations in 13% of the tumors were discovered, associated with, longer survival, regardless of behandeling.[40].

K-ras mutations in 21% of the tumors were discovered, were associated with significantly lower time to progression and survival time in patients treated with chemotherapy plus erlotinib. These results suggest that EGFR mutations may be a positive prognostic factor for survival might be independent of treatment with erlotinib, and that the combination of EGFR inhibition with chemotherapy, it should be better avoided in patients with K-ras mutations. Finally, it was reported that activation of Akt, determined by IHC and phosphorylation-specific antibodies, was associated with better response to gefitinib compared with the response in patients who had a negative score on P-Akt (phosphorylated Akt) .- There was a 844287992 statistically significant association between a positive P-Akt status and female gender, a history of non-smoking and histology of broncho-alveolar carcinoma, suggesting that activation of this pathway could be related to the presence of EGFR mutations. It should be noted that a determination of P-Akt in the absence of EGFR expression could signal transduction of EGFR by an independent pro-survival signal, which is a less effective anti-EGFR therapy could make.

In fact, a P-Akt-positive staining in 'EGFR-negative' NSCLC (ie tumors without mutation or change in the EGFR gene or protein with a low EGFR expression) predict a worse treatment outcome, [41], while positive staining P-Akt in 'EGFR-positive "cases (ie, tumors that contain a mutation, or a high number of gene copies or high levels of EGFR protein), a subgroup of patients who are particularly sensitive to gefitinib could identify. Together, these results indicate that the decision on treatment with anti-EGFR drugs ideally should be based on a combined use of different markers by different techniques should be established (eg, PCR-direct sequencing, IHC and FISH) with different types of tissue samples (eg, pieces of tissue removed and DNA).

Secondary Mutations in the EGFR gene and Acquired Resistance to EGFR Inhibitors.

In addition to primary resistance to anti-EGFR therapy is associated with the presence of a K-ras mutation [35], develops eventually resistance in most NSCLC patients who were or are refractory to gefitinib and erlotinib but EGFR mutations, which leads to progressive disease during treatment. Acquired resistance to EGFR inhibitors was found to correlate with the occurrence of an additional EGFR mutation. [42.43]. One study describes the occurrence of a secondary mutation in a patient with NSCLC following successful treatment with gefitinib [43]. While the first mutation, a deletion in exon 19, resulted in sensitivity to the treatment, half induced point mutation in exon 20, leading to the substitution of Thr790-to-Met led resistance to several EGFR inhibitors. Another group identified the same Thr790-to-Met mutation in two of five patients with acquired resistance to gefitinib or erlotinib [42], and the sixth patient whose tumor progressed in the adjuvant gefitinib after completeresection. The mutation could not be detected in untreated tumor samples. Biochemical analysis of transfected cells and studies of growth inhibition of cancer cell lines confirmed that the Thr790-to-Met mutation led to resistance in tumors with EGFR mutations that are usually sensitive to gefitinib or erlotinib [42]. Two reports from Asia showed that the Thr790-to-Met mutation may be present from diagnosis and resistance to EGFR TKIs bring about. [44.45]

These findings are very similar to the experience with imatinib in gastrointestinal stromal cell tumors and chronic myeloid leukemia , respectively, where secondary changes in the C-KIT and BCR-ABL gene were associated with a major cause of acquired resistance to imatinib. [46.47 ].

Interestingly, the EGFR protein that the second mutation, was sensitive to CL-387785, a specific and irreversible anilinoquinazoline TKI of EGFR, suggesting that the second generation TKIs of EGFR could play a role in the treatment of NSCLC. In this respect, clinical drugs developed to inhibit tumors that are resistant to gefitinib and erlotinib with two EGFR mutations (Leu858-to-Arg and Thr790-to-Met) in vitro. This study identified two drugs, EKB-569 and CI-1033, this broader spectrums for members of the ErbB family than erlotinib and gefitinib, and showed activity against resistant mutant.[48]. EGFR mutations are more common in East Asia, where the incidence exceeds 40%, and the higher prevalence of these mutations is correlated with a higher response. There have been reports of secondary mutations in Asians, but their role in changing the sensitivity to EGFR inhibitors remains unclear [18.49]. It has been reported that deletions in exon 19 could identify sensitive tumors more accurately than point changes, which were found in some patients whose tumors grew when treated with gefitinib. [18]. It is possible that some point changes that were discovered in exons 20 and 21 resistance rather than sensitivity to calls. Furthermore, predicts the presence of mutations in the region of the ATP-binding probably not the response to anti-EGFR therapy using monoclonal antibodies, such as cetuximab. [50.51]. Interestingly, recently reported that two patients responded to gefitinib after failure of several chemotherapy and cetuximab. [52] This finding could indicate that monoclonal antibodies and TKIs different mechanisms of action and could effectively be combined to their spectrum of activity broadening.

Selection of patients for treatment with EGFR Inhibitors

The relationship between EGFR mutations and particular patient characteristics including sensitivity to drugs, such as female gender, adenocarcinoma histology, never-smoking status, race and Asian, is striking. Still, a selection of patients for treatment with these drugs that is only based on the presence of EGFR mutations controversial, since not all patients benefit from treatment harboring a mutation, and some patients with mutations are resistant to TKIs. It might indicate that deletion mutants consistently lead to sensitivity than point mutations, which are also difficult to determine by direct sequencing. Unlike some disposal point mutations change the protein building effect in a manner that best prevents enzyme inhibition by EGFR TKIs. Moreover, other factors, including strengthening of the EGFR gene and the activity of molecules downstream of EGFR, such as P-Akt, and mutations of K-ras, may play a role in the definition of sensitivity to EGFR inhibitors. Based on available data, limiting treatment to patients who have an EGFR mutation can not be justified, although patients with mutations appear to be a very big chance to response to TKI therapy. The occurrence of secondary mutations that the composition of the ATP-binding pocket change should be examined, and the development of second-generation EGFR inhibitors that overcome this resistance is necessary.

There, prospective clinical trials with patients based on biological characteristics and clinical features are selected to be performed. At the moment it would be inappropriate for treatment with a TKI be excluded solely on the basis of the mutational status of EGFR. The establishment of patient characteristics,such as smoking status, gender and histology, would help the choice of therapy for patients with advanced NSCLC. First line treatment with an EGFR TKI in patients with EGFR mutations or in patients who never smoked are women and have adenocarcinoma histology, is certainly reasonable, either as single agent or in combination with chemotherapy. An EGFR TKI would be preferable in the second and third line therapy in patients with these characteristics, rather than chemotherapy, due to the lower toxicity profile and simple administration. Several studies that take account of EGFR mutations and clinical characteristics for the selection of patients have now been put under way or planned. Given the appearance of secondary mutations that confer resistance to TKIs, should clinical trials should try to repeat biopsies in an attempt to give direction to decision making in relation to the treatment.

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Sidebar: Glossary
In-Frame Deletions: Mutations That affects three nucleotides do not Disrupt the translational reading frame of the DNA sequence; Such Mutations can, however, lead to the synthesis of an abnormal protein product

Point Mutations: Mutations That Consist of the replacement (transition or trans version), Addition, or deletion (frame shift) or one nucleotide

Rna Interference: Use of double-stranded RNA to interfering with normal RNA processing, Causing rapid degradation of endogenous RNA and precluding translation, a simple method of studying the effect of absence of a gene product

Single-Strand Conformation Polymorphism: The use of electrophoresis to separate single-stranded nucleic acids based on subtle differences in Their DNA sequence, or at a single base pair, Which results in a differential structure and a secondary Measurable difference in mobility through a gel

Laser Capture Microdissection: A method for procure pure cells from specific microscopic regions of tissue sections, Which Help to automate and standardize microdissection

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Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, Netherlands e-mail from Dr. Giaccone

Giaccone G is Head of the Department of Medical Oncology, and JA Rodriguez is a member of the Department of Medical Oncology at the Free University Medical Center, Amsterdam, Netherlands

Competing Interests: The authors Declared They have no special destinations competing

Here the original article published by Medscape.

EGFR Inhibitors: What Have We Learned From the Treatment of Lung Cancer? Posted 11/22/2005

Giuseppe Giaccone, Jose Antonio Rodriguez

Summary
Tyrosine kinase inhibitors directed against the epidermal growth factor receptor (EGFR) are the first molecular-targeted agents to be approved in the U.S. and Other countries for the treatment of advanced non-small-cell lung cancer after failure of chemotherapy. Some Patient Characteristics, Such as never-smoking, female gender, East Asian origin, adenocarcinoma Histology, and bronchioloalveolar subtype, are associated with a Greater benefit from treatment with EGFR inhibitors. Recently, studies have Identified gene Mutations targeting the kinase domain of the EGFR That are related to the response to inhibitors. Most EGFR Mutations Predict a higher benefit from treatment compared with wild-type receptors and are correlated with clinical features related to better outcome, some EGFR Mutations, however, confer drug resistance. Usually the analysis of material available from lung cancer patients, Using Techniques Such As direct sequencing to determining EGFR mutational status, Can Be Technically challenging. In this Regard, high EGFR copy number and EGFR protein detected by immunohistochemistry CAN Also be Used to select patiënten Those who Would benefit from treatment. Prospective validation of biological and clinical markers of sensitivity needs to be Performed.

Introduction
Lung cancer is the leading cause of cancer-related death in the Western World and the mortality rate is Increasing Rapidly in Asia, 1.2 million cancer deaths worldwide from lung cancer Were in the year 2002. [1] Primarily There are two major types of lung cancer: non-small-cell lung cancer (NSCLC) and small-cell lung cancer. More than 50% of NSCLC patients are Candidates for treatment with systemic chemotherapy, Either for advanced disease, or as adjuvant or neoadjuvantTreatment in Addition to local therapy. Chemotherapy HAS, however, modest activity in NSCLC and in the Past few years, Several Drugs That are more specific for cancer cell targets have Shown activity in NSCLC. [2, 3] There are two molecular-targeted agents approved for the treatment of advanced NSCLC: gefitinib (Iressa ®, AstraZeneca, Wilmington, DE) and erlotinib (Tarceva ®, OSI Pharmaceuticals Inc., Melville, NY). Both agents are small molecules That Belong to the quinazolinamine class and inhibit the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) by competing with ATP for the ATP-binding site. [4] Besides theses two Rather selective tyrosine kinase inhibitors (TKIs ) or EGFR, other TKIs with a Broader spectrum of activity, and monoclonal antibodies to the extra cellular domain of the receptor are Also Being Tested in advanced NSCLC. [4] Among Broader spectrum TKIs are lapatinib and canertinib, Which have activity on more members of the ErbB family of receptors, and ZD6474 and AEE788, Which inhibit the vascular endothelial factor receptor in Addition to EGFR. After failure of chemotherapy, gefitinib and erlotinib are playable objectifying to induce major responses in approximately 10% of Caucasian and 25-30% of Japanese Patients Patients (gefitinib) with NSCLC tumors. The response rate to the EGFR monoclonal antibody cetuximab (Erbitux ®, ImClone Systems / Bristol-Myers Squibb) Similar Appears in The Same setting, but no experience is available in Asian patient. [4]

Biological Aspects of EGFR Mutations Associated With Tyrosine Kinase Inhibitor Responsiveness A major breakthrough in the field of EGFR-targeted therapy was seen in 2004 with the identification of Somatic Mutations in the EGFR gene, Which Were Closely associated with a Favorable clinical response to gefitinib and erlotinib treatment in NSCLC patients. [5,6,7] These genetic Alterations consisted of small in-frame deletions or POINT MUTATION in EGFR exons 18-24, Which encode the kinase domain of the protein and are clustered in two mutational hot spots in the EGFR gene. Alterations Were The Most frequently detected small deletions in exon 19 That Eliminate amino acids 747-750 (Leu-Arg-Glu-Ala), located around the active site of the kinase, and Point Mutations in exon 21 That resulted in the amino acid substitution Leu858Arg, a residue located in the activation loop. [5,6,7]

The in vitro analysis of the phenotypic effects associated with the expression of mutant EGFR proteins Provided information on the Molecular and Cellular Mechanisms Underlying the enhanced responsiveness to gefitinib and erlotinib in NSCLC tumors with patiënten That Have EGFR Mutations. The efficiency of drug synthesis was significantly higher in terms of blocking the activity of the receptor and Decreasing cell viability in cell lines harboring mutant EGFR Those genes. Transfection experiments in NSCLC-derived cell lines [5,8,9] revealed That, although mutant EGFR undergo more pronounced and sustained activation upon ligand binding Than the wild-type receptors, EGFR Their activity is more efficiently inhibited by gefitinib and erlotinib. [ 6,7,8] The molecular basis for Such differential sensitivity of mutant EGFR proteins Remains To Be Established, as wild-type and mutant receptors do not appearing to differ significantly In Their ability to interact with synthesis inhibitors in-vitro. 763 509 854 Importantly, binding of the ligand to the mutant EGFR was Shown to result in the selective activation of downstream signaling pathways That PromoteLimit cell survival, Such As the protein kinase B (Akt) and the signal transducers and activators of transcription (STAT) pathways. [8, 9] The pro-survival signals transduced by hypothesis downstream signaling molecules Appear to be essential for NSCLC cell That express mutant EGFR, as theses cells under went massive cell death after pharmacologic inhibition of the Akt and STAT signaling or RNA interference-mediated specific downregulation of the mutant receptor. [8] These observations Indicating That the Increased efficacy of gefitinib and erlotinib in NSCLC patients bearing EGFR Mutations Results, at least in part, from the Abrogation of the pro-survival signals on Which the tumor cells expressing a mutant receptor have become dependent. [8.11]

EGFR Mutational Status as a Predictive Marker of Tyrosine Kinase Inhibitor Response The initial studies assessed EGFR Mutations That patiënten selected with striking and long-lasting responses. [5.6] Mutations Were must frequently detected in a subpopulation of NSCLC patients with Characteristics associated with a better treatment outcome: women, non-smokers, patient, or Japanese origin, and patients with adenocarcinoma and Histology, in Particular, bronchioloalveolar carcinoma. [4.12] The Largest Database of Patients Treated Currently available with a single agent is from the BR. 21 study, a randomized trial where clause erlotinib was compared with best supportive care in patientwith advanced NSCLC who had failed one or two lines of chemotherapy. This study demonstrated significantly improved survival That erlotinib, although the response rate was only 8.8% 112,160,671 .- This modest response rate Might not be solely attributable to Such an important survival gain. It Should Be That noted a large proportioning of patients in this study had Either a minor response or stable disease; Considering That One of the major effects of EGFR inhibitors observed in preclinical studies was the ability to reducing cell proliferation, [14] Might this be indeed an important effect. In NSCLC cell lines, apoptosis was only observed Botha When the extra cellular signal-regulated kinase and the Akt pathways Were blocked by EGFR inhibition, [14,15] whereas in musts cell lines and only this was not Possible inhibition of proliferation was observed. Of the 731 patients included in the study BR.21, 197 Were assessed for EGFR status Using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and mutation analysis. In multivariate analysis, objective response was significantly associated with adenocarcinoma Histology, never-smoking status, and expression of EGFR by IHC.[13] Univariate analysis showed That survival was significantly longer in the erlotinib arm When EGFR expression was high and tumor cells had a high number of copies of EGFR genes, detected by FISH. However, The Influence of the EGFR status Determined By Any of the Three Methods Used by multivariate analysis was not significant. This study casts some Doubts on the validity of the mutational status of EGFR as a predictor of response and survival on larger Populations of Patients. In this study, however, a large number of Mutations Were Identified (24/45, 53%) That Were Previously unreported. These data, therefore, need Might Further confirmation, Perhaps by Carrying out a cross-validation analysis. The Functional Implications of the Novel Mutations Should Also be confirmed.

The proportioning of Patients who benefited from anti-EGFR therapy in this study[13] and Other large clinical studies [16.17] Exceeded the proportioning of NSCLC patients expected to harbor a mutant EGFR gene, based on the prevalence of EGFR Mutations Determined in the initial analysis. Approximately 40-50% of patients in two large Phase II trials of gefitinib derived clinical benefit, Which was similar to the number of Patients who Experienced a major response plus stable disease. [16.17] These observations Indicated That, although the presence of an EGFR mutation can-a Favorable reliably predict response to gefitinib and erlotinib treatment, a subset of Patients who benefit from theses drugs Would Would not be selected on the basis of EGFR mutational status.

The Close Relationship Between the Presence or mutant EGFR genes and the clinical response to TKIs Has Been CONFIRMED consistently (Table 1). However, as the number of NSCLC patients under going EGFR mutation analysis increases, it HAS become clearer That a small, but sizable, Subset of Patients with Mutations do not respond to TKI treatment. Conversely, it is evident That Also some patiënten who responded to gefitinib and erlotinib therapy have wild-type EGFR. These observations suggest That Might procedures for Evaluating EGFR mutational status need to be optimized, and Additional markers need to be Established to accurately identify all of the NSCLC patients Who can benefit from anti-EGFR therapy. Findings similar to gastrointestinal stromal tumors in patient with an indication That Mutations in the gene C-KIT, it Appears That Might differential EGFR Mutations confer differential sensitivities to TKIs in NSCLC. In two reports from Asia (Taiwan and Japan), or EGFR Mutations Where the incidence is much higher now in the Western world, Mutations in exon 19 of the EGFR gene to confer sensitivity more Appear Often Than Point Mutations in exons 20 and 21. [ 18.19] Given the relative infrequency of EGFR Mutations in Caucasians, it is Difficult at present to extend beyond thesis Findings Asian patiënten.

Optimizing EGFR Mutation Analysis
It Has Been That postulated the presence of undetected EGFR Mutations Might explain some of the Favorable responses to TKIs observed in NSCLC patients bearing seemingly wild-type EGFR. [12.20] Although Several of the NSCLC series Analyzed for the Presence of EGFR Mutations Consist , Wholly or in part, or frozen tissue samples (Especially from resected patient), The Most common type of sample available for analysis, DNA Extracted from Formalin-fixed, paraffin-embedded tissues. Axis a result of the fixation procedure, hypothesis Usually samples yield low-quality, degraded DNA That Can Be Difficult to analyze. On the otherhand, in patients with advanced lung cancer, or at only small diagnostic material is available, and it is Often cytology That makes it Difficult to Implement direct sequencing, Because of Insufficient number of tumor cells present in thespecimens. In Addition, wild type EGFR DNA sequences from the normal cells in the sample can-mask the presence of Mutations. In this Regard, there is some evidence thats the CPE must Used only method of polymerase chain reaction (PCR) amplification followed by direct sequencing Might not detect Mutations That Can Be Identified Using a differential analysis method, Such As single-strand Conformation POLYMORPHISM.[21] Further More, the analysis could-be improved by the use of novel techniques based on denaturing high-performance liquid chromatography, Which Appear to Be More Sensitive Than direct sequencing for the detection of EGFR Mutations, as well as by [22] Developing Methods to specifically determining the Mutations prevalent types of wine based on simple PCR and Restriction Enzyme Digestion. More over, novel data Indicating That the use of laser capture microdissection to select tumor cells Might lead to the identification of EGFR Mutations That are undetectable When The non-microdissected tumor tissue is analyzed.[23] However, most of These Additional procedures are Technically demanding thing and Might Be Difficult to Implement in many clinical laboratory settings. In summary, The Possibility That some EGFR Mutations Might Be Difficult to detect Using easy-to-Implement, Technically straightforward approaches Highlights The Importance of Establishing Additional predictive markers, in Particular Those That Would not depend on PCR-based analysis of DNA Extracted from tumor tissue.

Additional Markers to Predict Response to EGFR Tyrosine Kinase Inhibitors in NSCLC In otherwords molecular-targeted approaches to therapy, Such as the treatment of meta-static breast cancer with the anti-human epidermal growth factor receptor (anti-HER2) antibody trastuzumab (Herceptin ®, Genentech Inc.., San Francisco, CA), overexpression of the target protein (ie HER2) is a critical marker That is Used to select patiënten Who Will benefit from treatment TKIs. Using EGFR[24] In contrast, initial studies on NSCLC patients Indicated That gefitinib was no differential treatment outcome in patient who overexpressed Both HER2 and EGFR, compared with Those who had no HER2 overexpression. [25.26] Similar Results Were Obtained in the first phase II study of erlotinib.[27] In a more recent report, [28] EGFR status was Determined by Using Three Different Methods: Direct sequencing to Investigate the presence of Mutations, EGFR FISH gene copy number to Assess, Evaluate and IHC to EGFR protein expression levels. In this study, "EGFR positivity 'in any thesis or assays was significantly associated with a better response to gefitinib, but a better survival was found to be significantly associated only with a high EGFR gene copy number in multivariate analysis. Results These are, gene rally, in concordance with the Results Obtained in erlotinib-treated patients in the supporting BR.21 study.[29] Consistent Results Determining the predictive value of EGFR gene copy number in gefitinib-treated Patients Have Been Obtained in independent studies Using Either FISH 772204596 or a quantitative PCR[23] assay. interestingly, a significant correlation Between the Presence of EGFR Mutations and a high EGFR gene copy number was seen, [23,28,31] Suggesting That the mutant allele of the EGFR gene is selectively Amplified in tumors, As It Has Been observed in EGFR-mutant NSCLC cell lines. [9.23] Although Potentially more appealing, the Increased gene copy number was based on a six-category scoring system Using FISH analysis and an arbitrary cut-off value by reverse transcription PCR. Will These assays need validation in prospective studies and confirmation by Other Laboratories.

Members of the ErbB family of receptors (Other Than EGFR) That Might heterodimerize with the EGFR, HER2 Such As, Also have bone Investigated as potential predictive markers for anti-EGFR therapy. Similarly, signaling molecules That function downstream of the EGFR pathway Such As K-ras and Akt, s been implicated to function as predictive markers. Although the response to gefitinib Appears to be independent of HER2 levels, as Determined by IHC, the presence of somatic [25] Mutations That alter the structure of the protein HER2 kinase domain Has Been Reported Recently in NSCLC patients. [32.33] These Mutations Consist of small insertions and duplication That target HER2 analogous to a region or exon 19 deletions in the EGFR gene. Mutations HER2 Seem to be more frequent in patient with Clinical Characteristics of Patients similar to the subpopulation bearing EGFR Mutations but, remarkably, They never occure simultaneously with EGFR Mutations. The prevalence and functional relevance of HER2 Mutations Should Be Investigated Further to determining Their value as markers for anti-EGFR therapy.

Interestingly, Mutations in EGFR or HER2 genes s been found to be mutually exclusive with K-ras Mutations. [34,35,36,37] Further More, thepresence of K-ras Mutations is associated with a Lack of response to gefitinib treatment and erlotinib.[35] Therefore, the presence of a K-ras gene mutation, Which is detected in approximately 30% of NSCLC, [38] is Likely to constitute a Useful marker for Selecting patiënten Those Who Will not benefit from anti-EGFR therapy. In a large, randomized study of chemotherapy with or without erlotinib as first-line therapy for advanced NSCLC patients, [39]EGFR Mutations, detected in 13% of tumors, Were associated with longer survival irrespective of race treatment.[40]K Mutations, detected in 21 % of tumors, Were Decreased significantly associated with time to progression and survival in patiënten Treated with Erlotinib plus chemotherapy. These Results Suggest That Might Be a positive EGFR mutation Prognostic factors for survival, independent of treatment with erlotinib, and thats the combination of EGFR inhibition with chemotherapy Should be avoided in patient with K-ras Mutations. Finally, it was Reported That activation of Akt, Determined Using IHC and phosphorylation-specific antibodies was associated with a better response to gefitinib compared with the response in patients who Were negative for P-Akt (phosphorylated Akt) .- There was a 844287992 Between statistically significant association P-Akt positivity status and female gender, a history of never-smoking, with bronchioloalveolar carcinoma and Histology, Suggesting That the activation of this pathway Might Be associated with the presence of EGFR Mutations. Should it be noted, however, That detection of P-Akt in the absence of EGFR expression could-represents the transduction of an EGFR-independent pro-survival signaling, Which Might render anti-EGFR therapy less effective. In fact, P-Akt-positive staining in 'EGFR-negative' NSCLC (ie tumors Lacking mutation or amplification of the EGFR gene or expressing low levels of EGFR protein) could-treatment predictors of a worse outcome, whereas -844 287 992 P-Akt-positive staining in 'EGFR-positive "cases (ie, tumors containing a mutation, high gene copy number, or high EGFR protein levels) could-identify a subgroup of NSCLC patients to gefitinib Particularly Sensitive. Collectively, theses Results Indicating That the decision Regarding treatment with anti-EGFR agents Should Ideally be based on the combined use of markers Several That Would be Evaluated Using differential techniques (eg PCR-direct sequencing, IHC and FISH) to differentiate types or samples (eg Extracted DNA and tissue sections).

Secondary Mutations in the EGFR Gene and Acquired Resistance to EGFR Inhibitors in Addition to the primary resistance or anti-EGFR therapy associated with the presence of K-ras mutation, [35] Eventually resistance must developement in NSCLC patients who initially responded to gefitinib and erlotinib but harbor EGFR Mutations, leading to disease progression treatment consistently. Acquired resistance to EGFR inhibitors Has Been Shown to be associated with the occurrence of an Additional EGFR mutation. [42.43] One study described the occurrence of a secondary mutation in a patient with NSCLC following successful treatment. gefitinib[43] While the first mutation , a deletion in exon 19, conferred sensitivity to the treatment, a second point mutation in exon 20, leading to the substitution Thr790Met, Several induced resistance to EGFR inhibitors. Another group Identified The Same Thr790Met mutation in two or five Patients with acquired resistance to gefitinib or erlotinib-427 793 461 and a sixth patient Whose tumor progressed When Treated with adjuvant gefitinib after complete resection. The mutation Could not be detected in untreated tumor samples. Biochemical analysis of transfected cells and growth inhibition studies with lung cancer cell lines CONFIRMED thats the Thr790Met mutation conferred resistance in tumors with EGFR Mutations That are Usually sensitive to gefitinib or erlotinib.[42] Two reports from Asia demonstrated thats the Thr790Met mutation can-be present from diagnosis and confer resistance to EGFR TKIs. [44.45]

These Findings Closely resemble the experience with imatinib in gastrointestinal stromal cell tumors and chronic myeloid leukemia, where clause secondary Mutations in the C-KIT and BCR-ABL genes, respectively, s been associated with a major cause of acquired resistance to imatinib. [46, 47] interestingly, the EGFR protein bearing the second mutation was sensitive to CL-387, 785, a specific and irreversible anilinoquinazoline or EGFR TKI, Suggesting That second-generation EGFR TKIs or Might have a role in the treatment of NSCLC. In this respect, agents going under clinical development for inhibition of the gefitinib-resistant and erlotinib-resistant tumors with two EGFR Mutations (Leu858Arg and Thr790Met) Were screened in vitro. This screening Identified two drugs, EKB-569 and CI-1033, Which have spectra for ErbB family members Broader Than erlotinib and gefitinib, and showed activityAgainst the resistant mutant.[48]EGFR Mutations are much more frequent in East Asia, Where the incidence Exceed 40%, and the higher incidence of Mutations thesis is correlated with a higher response rate. Secondary Mutations Have Been Reported in Asians, but Their role in modifying sensitivity to EGFR inhibitors bone HAS not yet elucidated. [18.49] It Has Been Reported That Might deletions in exon 19 more accurately identify sensitive tumors Than Point Mutations, Which Were found Whose patiënten in some tumors progressed on treatment with gefitinib.[18] It Possible That is quite some Point Mutations detected in exons 20 and 21 confer resistance Might Rather Than sensitivity. Over more, the presence of Mutations in the ATP-binding site Probably does not predict response to anti-EGFR therapy Using monoclonal antibodies, cetuximab Such As. [50.51] interestingly, it HAS Recently Reported That leg two patiënten responded to gefitinib after failure or Several chemotherapy regimens and cetuximab. This finding could[52]-Indicating That monoclonal antibodies and TKIs have differential Mechanisms of action and Might Be effectively combined in order to broaden the