Lung Cancer Newsletter Spring 2007

I. Tarceva and EGFR Inhibitors

A. Response to Tarceva and Iressa- EGFR Mutation

Studies have shown that response to Iressa and Tarceva* correlates with the existence of a mutation in the epidermal growth factor receptor. (EGFR).  These drugs are EGFR inhibitors with the EGFR the target of recent medical research.  Lynch first found in 2004, "mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response." Lynch (1).  We now see:                    

"Targeting the epidermal growth factor receptor (EGFR) has played a central role in advancing NSCLC research initiatives, treatment, and patient outcome over the last several years. Novel targeted therapeutic agents, including the small-molecule tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib and the monoclonal antibody cetuximab, have been developed to interfere with EGFR signaling Soon after the TKIs were introduced, it became apparent that certain patient characteristics, including a never-smoking history, adenocarcinoma tumor histology, female gender, and East Asian origin, were correlated with improved response to the drugs and improved survival after treatment  One of the explanations for the improved outcome in these patient subsets was elucidated in 2004, when somatic mutations within the EGFR  gene itself were discovered   EGFR mutations are associated with both TKI response and prolonged survival and occur more frequently in the NSCLC patient subsets mentioned above . The mutations cause EGFR to transduce an intracellular antiapoptotic signal, upon which the cancer cells become "addicted" .  Administered EGFR TKIs interrupt the necessary survival signal, thus rendering the cell exquisitely susceptible to apoptosis."  Seequist (14).

The mutation is shown in a test pioneered at Harvard and Dana Farber and now commercially available. Lynch (1).   Let me first review the medicine behind these new drugs.

B. Overview of the EGFR

           1. Erb Family

EGFR or Erb 1 is a member of the ErbB family of RTKs,  which includes ERB2, ERB3,  and ERB4.   The EGFR gene is located at chromosomal region 7p12.      

           2.  The Tyrosine Kinase Family

EGFR is  a member of the  receptor tyrosine kinase (RTK) family.  These are called cell-surface receptors which receive and transmit signals for other cells.  "Phosphorylated tyrosines lead to the activation of the downstream pathways of EGFR thus resulting in cell proliferation, differentiation, migration/motility, protection from apoptosis (cell death), or angiogenesis (formation of tiny blood vessels which facilitates metastasis ."  (Sugio (16).  Cancer is really a communication malfunction among cells or defect in cell regulation. 

EGFR is normally found on the surface of epithelial cells and has been found to be commonly overexpressed in a variety of human malignancies  Different ligands can lead to EGFR activation and subsequent signal transduction, including the epidermal growth factor, the transforming growth factor , and neuregulins. After ligand binding to the extracellular receptor domain, EGFR undergoes homo- or heterodimerization and autophosphorylation of its intracellular tyrosine kinase domain. These autophosphorylation events trigger a cascade of downstream signals that ultimately result in an increase of cellular motility, proliferation and invasion, and a block of apoptosis, contributing to cancer development and progression Toschi (21).   

 3. Mutation

Cells have a long-string of commands.  A small deviation in one of these commands. observable only through detailed testing, may contribute to carcinogenic behavior.   After finding a series of mutations in patients who responded, scientists presented the hypothesis that response is connected with mutation.

            4. Mechanisms of Iressa

Lynch explains the mechanism of Iressa ( generic name gefitinib) :

Gefitinib targets the ATP cleft within the tyrosine kinase epidermal growth factor receptor (EGFR), which is overexpressed in 40 to 80 percent of non–small-cell lung cancers and many other epithelial cancers.  EGFR signaling is triggered by the binding of growth factors, such as epidermal growth factor (EGF), resulting in the dimerization of EGFR molecules or heterodimerization with other closely related receptors, such as HER2/neu.  Autophosphorylation and transphosphorylation of the receptors through their tyrosine kinase domains leads to the recruitment of downstream effectors and the activation of proliferative and cell-survival signals.  Lynch 1

Apoptosis or cell death is one method by which the body rids itself of malfunctioning or defective cells.  One method by which a malfunctioning  EGFR promotes cancer, is to send anti-apoptotic signals so that these defective cells do not undergo cell death but instead proliferate.

 C.  Response and the EGFR 

        1. Response Rates for those with the EGFR Mutation

Conventional chemotherapy response rates are generally in the 20-25% rates for non-small cell lung cancer patients.   However the response rate for patients with the mutation is much greater frequently exceeding 60%.   Han found response rate for patients with the mutation of 61.5%. (2).  One study found in an astounding 91% response among Asian non-smokers with the mutation:

Twenty-one patients harboring EGFR mutations were treated with gefitinib and were considered assessable for responses and adverse events. Nineteen patients with EGFR mutations achieved objective responses (three complete responses and 16 partial responses), resulting in an overall response rate of 90.5%. Yoshida (7)

Sharma just published a comprehensive overview on Tarceva and Iressa treatment and states,  "Most retrospective studies to date have reported that 50-80% of EGFR-mutant NSCLCs respond to gefitinib or erlotinib, and more recent studies from Asia, where EGFR mutant NSCLC is 2-3 times more prevalent, have reported responses in more than 75% of NSCLC cases with mutant EGFR." Sharma (5).

        2. Why Those with the Mutation Respond to Tarceva

Studies first showed that EGFR mutation played a substantial role in some lung cancers.  Scientists then found the EGFR drugs were able to interrupt that aberrant signal.

"EGFR mutations are associated with both TKI response and prolonged survival and occur more frequently in the NSCLC patient...  The mutations cause EGFR to transduce an intracellular antiapoptotic signal, upon which the cancer cells become "addicted."   Administered EGFR TKIs interrupt the necessary survival signal, thus rendering the cell exquisitely susceptible to apoptosis." Sequist (14)

Clearly those with the mutation have impressive partial and complete responses exceeding other treatments. It is not clear to what extent Iressa increases survival, and in which groups.

Most retrospective studies to date have reported that 50-80% of EGFR-mutant NSCLCs respond to gefitinib or erlotinib, and more recent studies from Asia, where EGFR mutant NSCLC is 2-3 times more prevalent, have reported responses in more than 75% of NSCLC cases with mutant EGFR.  Most significantly, although improvement in overall survival for the small fraction of EGFR-mutant cases treated with gefitinib or erlotinib has not reached statistical significance in US and European studies, this has been readily apparent in Asian studies with larger fractions of mutant cases. Taken together, the data suggest that a subset of cases, marked primarily by EGFR mutations and in some cases EGFR amplification, show dramatic responses to TKIs. These responses might occasionally be durable (that is, >3 years), but in most cases they only last for ~6-12 months before resistant disease recurs.

Additionally some responders may not have an EGFR mutation. Not all patients who carry an EGFR mutation respond to gefitinib and, conversely, there are patients who respond to gefitinib without an EGFR mutation, making somewhere complicating drug selection.  Sharma  (5).   Generally those who respond will have the mutation and be non-smokers or less frequently light former smokers.  Scientists are now dividing EGFR mutations to determine which predictor response and resistance.

Studies have shown some differences in the clinical outcomes that are associated with different mutations. For example, NSCLCs that harbour exon 19 deletion mutations seem to respond better to gefitinib and erlotinib than tumours with point mutations in exon 21, such as L858R.  Sharma (5). A The different classes of specific mutations may vary in their clinicopathological correlations, downstream signaling events, or responsiveness to TKIs. The two major classes of mutations, deletions in exon 19 or the L858R point mutation in exon 21, may result in differential autophosphorylation of specific phosphate residues, resulting in differences in downstream signaling.

D. Location and Prevalence of EGFR Mutations

Non-small cell lung cancer (NSCLC) mutations are concentrated in exons 18-21 of the EGFR.  Glioma,  another type of cancer, involve another part of the EGFR, the extra-cellular domain.  Lynch (1). 

Sharma explains the mutations locations in NSCLC :

Tumor somatic mutations in exons 18 through 21 of the tyrosine kinase domain of EGFR were described in 13 of 14 NSCLC patients who showed objective clinical responses to gefitinib monotherapy, whereas these mutations were not detected in tumors in 11 patients who did not respond... EGFR kinase domain mutations target four exons (18-21), which encode part of the tyrosine kinase domain (the entire kinase domain is encoded by exons 18-24) and are clustered around the ATP-binding pocket of the enzyme Sharma (5)

The most prevalent of EGFR kinase domain mutations, accounting for 45% of EGFR mutations in non-small-cell lung cancer (NSCLC), are in-frame deletions of exon 19, nested around the LREA string of amino-acids located between residues 747-750 of the EGFR polypeptide. Another recurrent mutation is the L858R substitution in exon 21, within the activation loop of EGFR, which comprises approximately 40-45% of EGFR mutations...About 90% of the mutations detected in EGFR are composed either of in-frame deletions in exon 19 or a specific missense mutation in exon 21 (L858R).  Sharma (5).

The question arises, can we further delineate predictors of response among these various exons. Which exons are likely to experience responses, which are less likely to respond, and what particular mutations will confer resistance to Iressa.

                1. Exon 18

Exon 18 is the nucleotide binding group and comprises only about 5% of mutations. Sharma (5).  The mutation involves substitution of G719 in the nucleotide-binding loop of exon 18,

                2. Exon 19

                       a. Prevalence

The most frequently EGFR mutations are L858R and deletions in exon 19.  These two mutations have been confirmed to be activating mutations and are extremely sensitive to gefitinib. (Iressa) Han, (2).  Patients with an exon 19 deletion had a significantly longer overall survival... There were also trends toward higher response rate (73% versus 50%) and improved time to progression (24 versus 10 months) for the patients with an exon 19 deletion. See Jackman (6).   6 patients had a deletion in exon 19 (3 E746-A750, 1 L747-A750, 1 L747-P751insP, and 1 E746... {All six} patients with a mutated EGFR responded to gefitinib (100%). Han (2).

        3. Exon 20

                     a. T790M Iressa-resistant mutation

Scientists have found a second mutation develops at exon 20 T790M which provides resistance to Iressa/Tarceva. This microspopic and small change at one exon can mean that Iressa or Tarceva will no longer be effective for many patients.

Despite dramatic responses in EGFR-mutant cases of NSCLC treated with gefitinib or erlotinib, the development of drug resistance within 6-12 months of the initiation of therapy greatly limits the ability of these drugs to significantly prolong patient survival. A deeper understanding of the molecular and cellular basis of this phenomenon is crucial to the future development of alternative therapies to overcome this resistance.

A single secondary mutation in EGFR exon 20, T790M, is present in a subset of EGFR-mutant tumours that recur after an initial response to gefitinib or erlotinib. Using allele-specific PCR, the T790M mutation is detectable in approximately 50% of patients with NSCLC who relapse after an initial response to TKIs, although in some cases the mutation seems to be underrepresented in the tumour cell genome relative to the total number of EGFR alleles. This suggests that T790M might either be present in only a subset of resistant cancer cells, or might be present only in a minority of copies of the EGFR gene in each tumour cell.  Sharma (5).

We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Pao (12) 

There may be a hereditary component in the development of this mutation:                                                        

                   
"mutations in EGFR identify a subset of non-small cell lung cancer that respond to tyrosine kinase inhibitors. Acquisition of drug resistance is linked to a specific secondary somatic mutation, EGFR T790M. Here we describe a family with multiple cases of non-small cell lung cancer associated with germline transmission of this mutation.  Four of six tumors analyzed showed a secondary somatic activating EGFR mutation, arising in cis with the germline EGFR mutation T790M.  These observations implicate altered EGFR signaling in genetic susceptibility to lung cancer."  Bell (15)

         b. Other characteristics 

Patients with T790M tended to be women, never smokers, and carrying deletion mutations, but the T790M was not associated with the duration of gefitinib administration.

         c. Treatment for the Cell Mutation

Studies of pan-inhibitors suggests that they may be able to inhibit aberrant cell-signaling by this mutation.  T790M mutations block binding of erlotinib and gefitinib to the receptor.  New irreversible EGFR bind to the mutated EGFR and have preclinical activity in such tumors.  Sharma writes,

One of the main challenges in the treatment of NSCLC is to design inhibitors that can overcome the steric interference to drug binding conferred by the T790M mutation. Irreversible inhibitors seem to show some promise in this regard . In most cases, irreversible inhibitors form a covalent bond with crucial cysteine residues C Cys797 within EGFR or Cys805 within ERBB2 C in the active site of the respective enzymes. Given the fact that only EGFR and ERBB2 (as opposed to ERBB4) have cysteines at these corresponding positions, irreversible ErbB inhibitors show very high specificity for EGFR and ERBB2. Previous studies from our laboratory have shown that the irreversible dual EGFR and ERBB2 inhibitors, HKI-272 (Ref. 136) and HKI-357 (Ref. 37), as well as the irreversible EGFR inhibitor EKB-569 (Ref. 137) were all able to overcome gefitinib resistance owing to T790M... other studies have shown that the irreversible EGFR inhibitor CL-387,785 (Ref. 139), and the irreversible pan-ErbB inhibitor CI-1033 (also known as canertinib) can overcome resistance to L858R-mutated EGFR harbouring the T790M resistance-conferring mutation, whereas the reversible EGFR and ERBB2 inhibitor GW-572016 (also known as lapatinib) was ineffective in this regard. Sharma 5.

Testing and identifying T790M mutation is becoming an important goal. Once the mutation is identified, continued use of Tarceva becomes questionable with pan-inhibitors now important alternatives.

                        d. Smoking status and Exon 20

Generally EGFR mutations have been found in non-smokers. Lynch (1). However, one researcher observed, A the exon 20 insertion mutations showed no bias for sex (seven in males versus eight in females) or smoking status (seven in smokers versus eight in never smokers). Shigematzu (11).

        3. Exon 21

A point deletion at exon 21 at L858R has been identified. A recurrent mutation is the L858R substitution in exon 21 within the activation loop of EGFR, which comprises approximately 40-45% of EGFR mutations.  Sharma (5).   "Paez  reported that the lung cancer cell line H3255 harboring the L858R mutation in EGFR exon 21 was also highly sensitive to gefitinib  and also that both Akt and ERK1/2 pathways in H3255 were highly susceptible to the inhibitory effect of gefitinib."  Ono 22. 

D. Other Predictors of Response to Iressa/Tarceva K-Ras Mutations and Iressa

"K-ras is a downstream mediator of EGFR-induced cell signaling, and ras mutations confer constitutive activation of the signaling pathways without EGFR activation. Growing evidence indicates that K-ras mutations are important in the development of lung carcinomas (19). Pao et al. (20) examined 60 lung adenocarcinoma patients and showed that K-ras mutations are associated with a lack of sensitivity to gefitinib or erlotinib. K-ras mutations seem to be resistant to EGFR-targeting agents and are reported to be mutually exclusive to EGFR or HER2 gene mutations." (Ono 22).

E. Use of Tarceva/Iressa for those without EGFR mutations

Lynch' s seminal study found that those who responded to Iressa had EGFR mutations. Typically those who are non-smokers and light-former smokers with adenocarcinoma. Can Iressa be used outside that target group, and can those without the EGFR mutation benefit from Iressa and Tarceva.

        1. Response to Iressa for those without the mutation

Beginning with Lynch, studies have generally found response concentrated within the EGFR mutation group. Lynch (1). Some studies have questioned whether Tarceva increases survival, partly because the studies have included smokers without the mutation who did not responds. Evidence showing a survival advantage for smokers without the mutation is questionable.

There is nonetheless some evidence supporting the use of Tarceva for smokers. Daniele wrote, A of the 35 patients without EGFR mutation, 14% responded to gefitinib.  Danieli (3)

F. Addressing Resistance and Irreversible Inhibitors

            1. Mechanisms of Resistance

Many patients who initially respond to Iressa develop resistant cells.   What is the mechanism by which cells become resistant to Tarceva. A study examined a primary resistant cell mutation, L858R, and found the resistance associated with the development of the Kras mutation:

Patients with pulmonary adenocarcinoma carrying the epidermal growth factor receptor (EGFR) mutation tend to display dramatic clinical response to treatment with the EGFR tyrosine kinase inhibitor gefitinib (Iressa). Unfortunately, in many cases the cancer cells eventually acquire resistance, and this limits the duration of efficacy. To gain insight into these acquired resistance mechanisms, we first prepared HEK293T cell line stably transfected with either wild-type (WT) or mutant (L858R) EGFR, and then expressed oncogenic K-Ras12V mutant in the latter transfectant....The cells expressing the EGFR-L858R were exquisitely sensitive (to Iressa). {However} 293T cells expressing both EGFR-L858R and oncogenic K-Ras were able to proliferate even in the presence of high concentration of gefitinib probably by inducing Erk1/2 activation. We also expressed K-Ras12V in the gefitinib-sensitive pulmonary adenocarcinoma cell line PC-9, which harbors an in-frame deletion in the EGFR gene. The activated K-Ras inhibited the effects of gefitinib treatment on cell growth, cell death induction and levels of phospho-Akt, as well as phospho-Erk. These data indicate that activated Ras could substitute most of the upstream EGFR signal, and are consistent with the hypothesis that mutational activation of targets immediately downstream from the EGFR could induce the secondary resistance to gefitinib in patients with lung cancer carrying EGFR mutation. Uchida (13).

1. Lynch, T. J. Specific Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib, The New England Journal of Medicine 2004, 350:2129-39

2. Han, Optimization of Patient Selection for Gefitinib in NonB Small Cell Lung Cancer byCombined Analysis of Epidermal Growth Factor Receptor Mutation, K-ras Mutation, and Akt Phosphorylation, Clinical Cancer Research Vol. 12, 2538-2544, April 15, 2006

3. Daniele, Predicting gefitinib responsiveness in lung cancer by fluorescence in situ hybridization/chromogenic in situ hybridization analysis of EGFR and HER2 in biopsy and cytology specimens, Mol Cancer Ther 2007;6(4):1223B 9.

4. Eberhard, Mutations in the Epidermal Growth Factor Receptor and in KRAS Are Predictive and Prognostic Indicators in Patients With NonB Small-Cell Lung Cancer Treated With Chemotherapy Alone and in Combination With Erlotinib, Journal of Clinical Oncology, Vol 23, No 25 (September 1), 2005: pp. 5900-5909, Journal of Clinical Oncology, Vol 23, No 25 (September 1), 2005: pp. 5900-5909

8. Tang, EGFR Tyrosine Kinase Domain Mutations Are Detected in Histologically Normal Respiratory Epithelium in Lung Cancer Patients, Cancer Res 2005; 65: (17). September 1, 2005

13. Uchida, Activation of downstream epidermal growth factor receptor (EGFR) signaling provides gefitinib-resistance in cells carrying EGFR mutation, Cancer Sci 2007; 98: 357B 363

14. Sequeist , Response to Treatment and Survival of Patients with Non-Small Cell Lung Cancer Undergoing Somatic EGFR Mutation Testing, The Oncologist, Vol. 12, No. 1, 90-98, January 2007

15. Bell, Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFRNature Genetics 37, 1315 - 1316 (2005)
 
16. Sugio, Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking, British Journal of Cancer (2006) 94, 896-903.

17.  Sasaki, EGFR Mutation Status in Japanese Lung Cancer Patients: Genotyping Analysis Using Light Cycler Linical Cancer Research Vol. 11, 2924-2929, April 15, 2005

18. Riley, Update on Epidermal Growth Factor Receptor Mutations in Non-Small Cell Lung Cancer
Clin. Cancer Res., December 15, 2006; 12(24): 7232 - 7241.

19.  Horiike, Detection of Epidermal Growth Factor Receptor Mutation in Transbronchial Needle Aspirates of Non-Small Cell Lung Cancer . 2007; 131:1628-1634. 

20.  Riely, Clinical Course of Patients with Non–Small Cell Lung Cancer and Epidermal Growth Factor Receptor Exon 19 and Exon 21 Mutations Treated with Gefitinib or Erlotinib, Clinical Cancer Research Vol. 12, 839-844, February 2006.

21.  Toschi, Understanding the New Genetics of Responsiveness to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors
The Oncologist, Vol. 12, No. 2, 211-220, February 2007

22.  Ono, Molecular Mechanisms of Epidermal Growth Factor Receptor (EGFR) Activation and Response to Gefitinib and Other EGFR-Targeting Drugs,  Clinical Cancer Research Vol. 12, 7242-7251, December 15, 2006

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keywords, Tarceva, Iressa, clinical trial, response rate, partial response, Exon 18, Exon 19, exon 20, exon 21, resistance to Tarceva, studies on Tarceva, EGFR, epidermal growth factor inhibitors, tyrosine kinase inhibitors, Lynch, tyrosine kinase inhibitor, Erb 1, Erb group, epidermal growth factor inhibitor, new treatment for lung cancer .