Prognostic Impact of [18F]Fluorothymidine and [18F]Fluoro-D-Glucose Baseline Uptakes in Patients with Lung Cancer Treated First-Line with Erlotinib

3′-deoxy-3′-[18F]fluoro-L-thymidine (FLT) and 2′-deoxy-2′-[18F]fluoro-D-glucose (FDG) are used to visualize proliferative and metabolic activity of tumors. In this study we aimed at evaluating the prognostic value of FLT and FDG uptake measured by positron emission tomography (PET) in patients with metastatic non-small cell lung cancer (NSCLC) prior to systemic therapy with erlotinib. FLT and FDG maximum standardized uptake (SUVmax) values per patient were analyzed in 40 chemotherapy naive patients with advanced NSCLC (stage IV) before treatment with erlotinib. Prior therapy median SUVmax was 6.6 for FDG and 3.0 for FLT, respectively. In univariate analysis, patients with an FDG SUVmax <6.6 had a significantly better overall survival (16.3 months [95% confidence interval [CI] 7.1–25.4 months]) compared to patients with an FDG SUVmax ≥6.6 (3.1 months [95% CI 0.6–5.5 months]) (p<0.001, log rank). Similarly, low FLT uptake (SUVmax <3.0) was associated with significantly longer survival (10.3 months (0–23.3 months, 95% CI) compared to high FLT uptake (3.4 months (0–8.1 months, 95% CI) (p = 0.027). The independent prognostic value of baseline FDG uptake was demonstrated in multivariate analysis (p = 0.05, Cox regression). These data suggest that baseline SUVmax values for both FDG and FLT PET might be further developed as markers for prognostic stratification of patients in advanced NSCLC treated with tyrosine kinase inhibitors (TKI) directed against the epidermal growth factor receptor (EGFR). Trial Registration Clinicaltrials.gov, Identifier: NCT00568841


Declaration by the trial investigators
In January 1997, the international conference on harmonization issued the "Note for Guidance on Good Clinical Practice" (ICH-GCP). The studies of the LCGC conform, regarding planning, execution and analysis, to the basic principles of GCP, adapted to the requirements and conditions applying to quality assurance protocols. The studies adhere to the declaration of Helsinki.
The principal investigator and co-investigators undertake to adhere to the declaration of Helsinki and the basic principles of GCP and undertake to publish the results of the trials following completion of data analyses. After the 6 week treatment period with erlotinib it is recommended to continue erlotinib treatment in patients with clinical benefit (CR,PR,SD) until progression and to start standard chemotherapy in patients with progressive disease and a good overall performance state.

Duration of Treatment
Erlotinib treatment for 6 weeks followed by response assessment and continuation of erlotinib treatment in patients with clinical response and stable disease. • Concurrent systemic immune therapy, chemotherapy or therapy with any anticancer drug not indicated in the study protocol • Any investigational agent(s) within 4 weeks prior to study entry • Previous administration of any EGFR-targeted therapy (antibodies, small molecules and others) • Any medical, mental or psychological condition which in the opinion of the investigator would not permit the patient to complete the study or understand the patient information

Systemic treatment options in NSCLC
Non small cell lung cancer (NSCLC) is the leading cause of cancer death in the western world (Jemal et al., 2004). While only a minority of patients with early stage disease is cured by radical surgery, most patients present with locally advanced or metastatic disease (stage IIIB, stage IV). None of these patients will be cured and the overwhelming majority dies within the first year after diagnosis. Chemotherapy has been shown to increase survival and to improve quality of life in these patients. However, the increase in median survival time is marginal, i.e. in the range from 2 to 4 months corresponding to an increase in the 1 year survival rate in the range from 10 -20% (Spira and Ettinger, 2004). In patients with a good performance state the administration of a standard two drug chemotherapy regimen, not necessarily being platinum-based (D'Addario et al., 2005), is considered standard treatment. No significant differences could be observed between several of such standard regimens (Schiller et al., 2002). Similar effects, however, have also been demonstrated in older patients with monochemotherapy alone. Recently, in a large randomized trial the addition of the monoclonal anti-VEGF antibody bevacizumab to paclitaxel/carboplatin first line chemotherapy has been shown to cause an overall survival benefit of two months in non squamous cell NSCLC (Sandler, 2005). Nearly all patients suffer from relapse of the disease with a median survival time of only a few months following diagnosis of relapse. Several chemotherapeutic agents, among others docetaxel, gemcitabine, vinorelbine, exert therapeutic activity in these patients and even a moderate effect on survival compared to best supportive care could be shown for docetaxel (Shepherd et al., 2000). In addition, the new multitargeted antifolat pemetrexed exerts a therapeutic activity comparable to docetaxel, however, with a better tolerability (Hanna et al., 2004).

EGFR-targeted therapy in NSCLC
The disappointing results of chemotherapy stimulated an intense search for new therapeutics in NSCLC. The knowledge of the particular changes in molecular signaling pathways involved in malignant transformation and tumor progression has led to the generation of novel drugs ("biologicals") that specifically target at signaling intermediates of oncogenic pathways (e.g. small-molecule inhibitors of tyrosine kinases) or growth factor receptors on the cell surface (e.g. monoclonal antibodies). Several of these drugs have already been established in other solid tumors. Well-known examples are the monoclonal antibody (Mab) trastuzumab targeting HER2 in HER2-expressing breast cancer (Cobleigh et al., 1999) and the tyrosine kinase inhibitior (TKI) imatinib mesylate in chronic myelogenous leukaemia (Druker et al., 2001). In NSCLC the epidermal growth factor receptor (EGFR) represents an attractive target for such innovative drugs. The EGFR (HER1) is a member of the HER growth factor receptor family of tyrosine kinases. Activation of this receptor tyrosine kinase activates an intracellular signal transduction cascade with effects on cell growth, survival, angiogenesis and differentiation. The EGFR pathway is thus regarded as pivotal for the regulation of malignant growth in several cancers, among them NSCLC (Yarden, 2001). Three EGFR-targeted "biologicals" have already shown therapeutic benefit in NSCLC, i.e. one monoclonal antibody and two small-molecule tyrosine kinase inhibitors. The Mab cetuximab (Erbitux R ), which recently has received FDA approval for treatment of relapsed colorectal cancer, has been shown to improve response rates in NSCLC when added to platinum-based chemotherapy (Gatzemeier, 2003). The two oral TKI gefitinib (Iressa R ) and erlotinib (Tarceva R ) induce clinical responses in about 10% of relapsed NSCLC patients and stable disease in about 40% (Fukuoka et al., 2003;Kris et al., 2003;Shepherd et al., 2005). Chemotherapy-typical toxicity is avoided with acne-like skin lesions and diarrhoe representing the most prominent side effects. Recently, in a phase III multicenter trial of the National Cancer Institute of Canada a survival benefit was reported for relapsed NSCLC patients treated with erlotinib with a median survival time of 6.7 months (placebo: 4.7 months; . By comparison, despite impressive clinical responses in a subset of patients, no overall survival benefit in patients with relapsed NSCLC could be shown for gefitinib compared to placebo in a recently published randomized trial (Barge, 2004). Gefitinib had already received approval for the treatment of NSCLC patients after two chemotherapeutic treatment regimens in the USA, Japan, Australia and Switzerland due to the impressive clinical responses. But, until the final analysis of the data of the ISEL trial and further ongoing trials the administration of gefitinib is no more recommended (Barge, 2004). In the US and in the EU, erlotinib is approved for therapy of relapsed NSCLC after failure of at least one chemotherapy. Recently, first-line treatment of NSCLC patients in advanced stage (IIIB/IV) with erlotinib was evaluated in a phase II study and impressive clinical responses were reported. The non-progression rate was 55%. Best response was: 1 CR, 12 PR, 16 SD, 17 PD, 7 NE in this cohort (Giaccone, 2005). Furthermore, Jackman and colleagues performed a study in elderly patients with erlotinib monotherapy as a first line treatment. Efficacy was demonstrated with median 1 year survival rate of 46% and a median overall survival of 10.9 months comparable to results expected in the firstline treatment with combined platinum based chemotherapy (Jackman et al., 2007) (Schiller et al., 2002). In a German study using gefitinib monotherapy in the first line setting the progression free survival was described as 7 weeks (Reck et al., 2006). Further studies to test erlotinib in the line setting progression free survival are ongoing.

Correlation of EGFR/ KRAS mutations and EGFR immunohistochemistry to clinical benefit
Mutations in the EGFR (frequency 2-25%) correlate with response (partial response, complete response) to EGFR-TKI (Lynch et al., 2004;Paez et al., 2004) in patients with NSCLC. These mutations were found within the kinase-domain-encoding region of the EGFR gene in tumor DNA of erlotinib and gefitinib responders, but not in that of non-responders. These results suggest that the kinase domain mutations may lead to enhanced binding of erlotinib and gefitinib in the ATP-binding pocket of the kinase domain, and thus, to enhanced inhibition of the tyrosine kinase and, ultimately, to enhanced tumor control. The kinase domain of the EGFR gene is encoded by seven exons and can reliably be detected in the tumor tissue only when the tumor-cell population represents more than ~70% of the affected tissue.
Protein expression of EGFR assessed by immunohistochemistry has been demonstrated to be predictive for clinical response to treatment with either gefitinib or erlotinib (Cappuzzo et al., 2005;Shepherd et al., 2005).
Interestingly, there is a group of patients without mutations within the EGFR also responding to inhibition with EGFR-TKI (Pao et al., 2004). In addition, a substantial proportion of patients treated with EGFR-TKI experience a clinical benefit by achieving a stable disease state which is not correlated to EGFR mutations. Moreover, in the randomized trial (Tarceva vs. placebo) the survival benefit for patients with Tarceva was not correlated to EGFR mutational status Tsao, 2005). Thus, although EGFR mutations are highly correlated to response to EGFR-TKI, they do not predict clinical benefit and survival.
Mutations within KRAS have a negative prognostic impact on clinical outcome in NSCLC (Rodenhuis and Slebos, 1990). Activating mutations within the KRAS gene are found in 15-50% of lung adenocarcinoma and are mostly localized within exon 2 (Rodenhuis et al., 1988;Suzuki et al., 1990). It has recently been shown that mutations within KRAS confer resistance to the treatment with erlotinib and gefitinib and are mutually exclusive with EGFR mutations (Pao et al., 2005). Based on their low frequency of about 15% and a 50-60% progressive disease rate it is well conceivable that KRAS mutations do not represent the only determinant of resistance to erlotinib respectively gefitinib treatment.
Taken together, sequence analyses of EGFR or KRAS for pre-treatment prediction of clinical response will likely miss a subset of responders that might have experienced substantial clinical benefit from EGFR-targeted therapy (i.e. non-mutated responders). Further the mutational status of those patients with stable disease is not yet clear. Moreover, these analyses are presumed to be only rarely applicable in clinical routine diagnostic procedures. So far, no assays are available that identify those patients definitely not profiting from EGFR-TKI treatment, i.e patients with progressive disease under treatment.

FDG-and FLT-PET for assessment of response to treatment
The addition of FDG (fluoro-deoxy-glucose)-PET to conventional pre-treatment workup has now become part of the guidelines of the American Society of Clinical Oncology (ASCO) for the diagnostics of patients with lung cancer (Pfister et al., 2004). This is due to an increase in specificity and sensitivity as compared to conventional CT scans (Pieterman et al., 2000;van Tinteren et al., 2002). In addition, substantial progress in measurement of tumor cell proliferation by PET has been made by synthesis of FLT (fluoro-L-thymidine, (Shields et al., 1998). FLT is trapped in tissue after phosphorylation by thymidine kinase 1 (TK1), resulting in high accumulation in tumors compared to non malignant tissue. In NSCLC, a high correlation was found between standardized uptake value (SUV) measures of FLT uptake and Ki-67 scores, an established marker of tumor cell proliferation (Buck et al., 2003;Buck et al., 2002). In these studies, the specificity was 100% and the sensitivity was ~85%. These findings suggest FLT-PET to become an ideal complementary technology to FDG-PET due to its higher specificity. Here, we hypothesize that a decrease in tumor cell proliferation as an early event during the response to erlotinib may be visualized by FDG-and/or FLT-PET and, vice versa, absence of this response may predict early progress. This hypothesis is mainly built on the following findings: Tumor cell proliferation was rapidly reduced by erlotinib in an in-vitro model of lung adenocarcinoma (Pao et al., 2004). Since at least FLT-PET was shown to directly correlate to tumor cell proliferation (Buck et al., 2003;Buck et al., 2002), we assume that FDG-/FLT-PET will allow to early determine response status of a patient under erlotinib treatment. Further clinical and experimental data support our hypothesis: In several early clinical studies it has been demonstrated for different malignancies such as breast cancer (Rousseau, 2005), soft tissue sarcoma (Schuetze, 2005), glioblastoma (Charnley NG, 2005), rectum cancer (Di Fabio, 2005) and lung cancer (Griesinger, 2005;Schmuecking, 2005) that clinical response to chemotherapy can be detected by FDG-PET after 4-8 weeks. Sensitivity for response prediction outperformed conventional imaging in these studies. In squamous cell cancer of head and neck it has been demonstrated that FDG-PET after completion of treatment with an anti-EGFR antibody depicts response consistently (L Bastholt, 2005). Furthermore, not only response to treatment, but also clinical progression can be detected reliably by FDG-PET in mesothelioma (Kimura, 2005). In an in vivo model of fibrosarcoma Leyton and colleagues detected a decrease in FLT uptake as early as 24h after start of chemotherapy with cisplatin. In this model FLT outperformed FDG as tracer (Barthel et al., 2003;Leyton et al., 2005). Similar data were obtained in an allograft mouse model of a squamous cell tumor as well as an cervical adenocarcinoma model (Sugiyama, 2004). Moreover, in recent studies FDG-PET was shown to reliably predict early response of patients with gastrointestinal stromal tumors (GIST) to the small molecule tyrosine kinase inhibitor imatinib mesylate or SU11248 (Jager et al., 2004;Stroobants et al., 2003;van den Abbeele, 2005) on day 8. Also response of GIST to treatment with the mTOR inhibitor AP23573 can be detected by FDG-PET already on day 5 of treatment (Sankhala, 2005).

AIM OF TRIAL
Based on the survival benefit demonstrated in patients with relapsed NSCLC  as well as on its favourable toxicity profile compared to chemotherapy the EGFR-TKI erlotinib represents an attractive candidate to be included also in protocols for the first line treatment of NSCLC. Accordingly, preliminary data from phase II trials show promising response rates for erlotinib treatment in the first line setting (Giaccone, 2005). In parallel, in the near future further targeted drugs will be available for NSCLC treatment. For instance, the addition of the anti-VEGF monoclonal antibody bevacizumab has recently shown to prolong survival when added to standard chemotherapy as first line treatment of NSCLC. Thus, in order to optimize the choice and the sequence of new drugs in NSCLC treatment, an early identification of patients with no clinical benefit, i.e. patients with progressive disease under treatment, is desirable. In the ERLOPET trial, we aim at establishing PET analysis as a tool for early identification of NSCLC patients with no clinical benefit from monotherapy with erlotinib as first line treatment. A pre-treatment computed tomography (CT) scan will be performed for evaluation of the patients´ individual tumor burden. FDG-and FLT-PET scans will be performed between d -10 and d1 (but prior to therapy) for identification of pretreatment PET status. Between d7 and d14, FDG-and FLT-PET scans will be performed for the early identification of erlotinib non responders. The imaging procedures will be performed at different days, i. e. one procedure per day. After six weeks of treatment, final response evaluation will be performed using CT, FDG-and FLT-PET scans. These results will be correlated to the mutational status of the EGFR -and the KRAS gene and the expression of EGFR in pretreatment samples.

Trial design, primary and secondary objectives Design
Phase II study (therapeutic exploratory), single center, single arm, open-label

Primary Objective
To evaluate the accuracy of FDG-/FLT-PET analyses for early prediction of erlotinib nonresponse in patients with stage IIIB/IV NSCLC (measured by ROC analysis of the SUV) treated with erlotinib

Secondary Objectives
1. To identify PET characteristics for progressive disease following erlotinib treatment (changes in SUV during treatment) 2. To identify PET characteristics for response to erlotinib (changes in SUV during treatment) 3. To identify PET characteristics for stable disease following erlotinib treatment (changes in SUV during treatment) 4. To confirm the statistical association of EGFR mutational status and clinical response in NSCLC patients treated with erlotinib 5. To confirm the statistical association of KRAS mutational status and clinical resistance to treatment of NSCLC patients with erlotinib 6. To determine the discriminatory capability of FDG-/FLT-PET regarding EGFR and KRAS mutational status in NSCLC patients 7. To compare prognostic value of FDG-PET, FLT-PET and EGFR mutational status regarding clinical response in NSCLC patients treated with erlotinib 8. To identify EGFR and KRAS sequence characteristics for clinical response and stable disease following erlotinib treatment 9. To assess safety of Erlotinib 10. To determine response rates 11. To determine rates of stable diseases 12. To determine one-year FFTF 13. To determine one-year OS 14. To determine median overall survival time

Recruitment
It is planned to recruit a maximum of 40 patients over a 15 months period.
Patients will be recruited after screening only if all inclusion criteria are met, no exclusion criteria are met and written informed consent has been obtained. No difference in recruitment concerning gender will be applied. According to the distribution of NSCLC more male than female study participants are expected.

Duration of the study
The expected duration from beginning of recruitment to final data analysis will be 33 months (15 months recruitment, 15 months treatment (6 weeks for each patient, further treatment after 6 weeks is not part of the study), 18 months follow-up).

Termination of protocol treatment
The patient can discontinue trial participation at his/her own wish at any time. Protocol treatment can be terminated based upon the decision of the treating physician, due to unacceptable toxicity, progression, serious intercurrent disease or pregnancy. In any case, following a formal or an actual termination, the patient's follow up should be documented.

Early termination of trial
The trial can be terminated early by the principal investigator in consultation with the coinvestigators, if the formal termination criterion is reached or if patients' safety appears to be at risk. In case of termination of the trial the review board will be informed.

Pathological diagnosis
Diagnosis of NSCLC will be made/confirmed in the Institute for Pathology, University Clinic Cologne.

Procurement of biopsy material for biological analyses
In case of study inclusion material that is not required for histological diagnosis will be stored under the respective conditions (room temperature for paraffin-embedded tissue, -80° C in a phone-alarm secured freezer in a local tissue bank of MTBTI in case of frozen or fresh material).
The following persons are responsible for the safe processing and storage of all biological material obtained during the study:

Pseudonymization
After inclusion into the trial each patient will be given a consecutive number. All documents (CRF) will be marked only with this number. Personal data and anonymous number will be stored separately.

Mutation analysis
Processing of tissue will be performed according to standard operating procedures (SOPs) established in MTBTI. Pretreatment biopsy material (tumor purity >70%, alternatively: tumor cell enrichment by microdissection) will be analyzed for EGFR mutational status by exon sequencing. A PCR has been established that allows for amplification and direct sequencing of EGFR mutational hotspots in exons 18, 19 and 21 from paraffin-embedded tumor tissue. Further a PCR amplification of KRAS exon 2 was established in the MTBTI (Zander et al. in preparation). In control experiments that were carried out on non-selected paraffinembedded lung cancer specimens from a local tissue bank, PCR efficiency for all target amplicons was 100%. Target sequences will be amplified by single-round PCR. Purified PCR products will be bi-directionally sequenced (sequences controlled for bi-allelic target amplification; quality threshold: Phred score >30). Electropherograms will be manually and computationally (software: DNAsis) compared to germline sequences. In those tumors where tumor cell purity is estimated to be <70%, tumor cells will be enriched by laser-assisted microdissection (PALM system, available through a close collaboration with the Institute of Pathology, University of Cologne). All technology is established in the MTBTI laboratories and there is extensive experience with generating and evaluating molecular genetic data.

EGFR immunohistochemistry
A positive correlation between EGFR positivity in immunohistochemistry and response to therapy has been demonstrated in relapsed NSCLC patients treated with erlotinib in the BR.21 phase III trial . Therefore, EGFR expression in primary paraffin embedded tumor tissue will be analyzed. EGFR staining will be performed in the Institute of Pathology of the University Hospital Cologne following standardized procedures established for clinical application.

Pre-Study Screening (staging examination)
Within a maximum of 14 days prior to registration, all patients will be screened by the investigator. Each patient must provide a written informed consent to the trial procedures. The patient must be informed about the study verbally and by the patient information by the principal investigator or one of the co-investigators, before informed consent is obtained. After obtaining written informed consent, the patient will be screened for eligibility. The following will be assessed: •

Patient preparation
For FDG-PET patients must refrain from ingestion for 12 hours prior to the PET examination; only the ingestion of carbohydrate-free drinks is permitted. The current glucose level should be measured before starting PET. Fasting glucose level over 130 mg/dl or known diabetes mellitus are exclusion criteria. Optionally regular insulin (e.g. 2 units) may be administered if blood sugars are unexpectedly high, provided that monitoring and emergency care are assured. For FLT-PET no special preparation is necessary.

Administration of tracers, scanning and data evaluation
18F-FDG and 18F-FLT will be synthesized at the Max-Planck-Institute for Neurological Research at the University of Cologne following standard procedures. A dose of 370 MBq FDG and 300 MBq FLT will be injected intravenously for whole-body examination by PET. Scanning will commence ~45-60 minutes after injection of tracers. PET instruments with fullring detectors will be applied. Emission data will be corrected for attenuation, scatter and random and will be reconstructed to plains of 3.125 mm width and 128x128 voxels. For quantification of tracer uptake, standard methods, such as standardized uptake values (SUV) will be applied.

46,3 mSv (m)
The used dose is the minimal dose to obtain high quality data suitable for further analysis

Patient briefing and termination of protocol treatment
After completion of staging, the patient is informed about the trial, including the following aspects: title and aim of the trial, nature of the treatment, side effects, reason for recruitment, passing on of data and material samples, insurance, the ethics committee vote and the patient's freedom to decide. The patient receives an information brochure and an informed consent form.
The signed original consent form and the record of the briefing remain in the patient's files. The patient will receive a copy of each document.
The patient can make a free decision on participation in the trial at any time. If trial participation is refused, the treatment can be freely chosen in accordance with the treating physician.
Termination of protocol treatment can occur at the patient's wish or if the treating physician decides to terminate it due to unacceptable toxicity, serious concurrent disease or pregnancy. Documentation after termination will be performed

Informed consent
The investigator assumes the responsibility of obtaining written informed consent for each patient or the patient's legally authorized representative before any trial-specific procedures are performed and before any trial drug is administered.
Patients meeting the criteria set forth in the protocol will be offered the opportunity to participate in the trial. To avoid introduction of bias, the investigator must exercise no selectivity with regard to offering eligible patients the opportunity to participate in the trial.
Patients or parents/legal guardians of all candidate patients will receive a comprehensive explanation of the proposed treatment, including the nature of the therapy, alternative therapies available, any known previously experienced adverse reactions, the investigational status of the trial drug, and other factors that are part of obtaining a proper informed consent.
Patients will be given the opportunity to ask questions concerning the trial, and adequate time to consider their decision to or not to participate.
Informed consent will be documented by the use of a written consent form that includes all the elements required by regulations and ICH guidelines. The form is to be signed and dated by the patient or patient's legally authorized representative and by the person who administers the consent process. A copy of the signed form will be given to the person who signed it, the original signed consent form will be filed with the patient's medical records. The date and time of the informed consent must be recorded in the source documents.
If an amendment to the protocol changes the patient participation schedule in scope or activity, or increases the potential risk to the patient, the informed consent document must be revised, submitted to the EC for review and approved by the EC before use. The revised informed consent document must be used to obtain re-consent from any patients currently enrolled in the trial if the patient is affected by the amendment, and must be used to document consent from any new patients enrolled after the approval date of the amendment.

Patient privacy
The investigator affirms and upholds the principle of the patient's right to privacy. The investigators shall comply with applicable privacy laws.

Confidentiality
The written informed consent form will explain that the trial data will be stored in a computer database, maintaining confidentiality in accordance with local data legislation and the EU directive 95/46/EG. Subjects in this database will be identified by patient identifier only. The patient information will also explain that for data verification purposes, authorized representatives of the sponsor, a regulatory authority, an ethics committee, or an institutional review board may require direct access to parts of the medical records relevant to the trial, including subjects' medical history.
Especially, anonymity of the patients will be guaranteed when presenting the data at scientific meetings or publishing them in scientific journals.

Reporting and recruitment
If after completion of staging the patient is qualified for the trial and he/she gives written informed consent, recruitment will be performed by LCGC in Köln. For recruitment, the following is required: • all entry criteria satisfied; • no exclusion criterion satisfied; • staging procedures completed according to protocol.
Patients should be asked to agree to be contacted by the trials coordination center.
This agreement and the patient's address are to be recorded on the REG form.
Patients who wish to avoid sending their personal data to the trials coordination center may be enrolled anonymously under a pseudonym code. Prerequisite for anonymous enrollment is, however, commitment to follow-up and documentation.
Three phase-II single agent studies with erlotinib at 150 mg daily dose were performed (I) in patients with chemotherapy resistant, HER1/EGFR-expressing NSCLC of all histological subentities (Perez-Soler et al., 2004), (II) in patients with bronchoalveolar carcinoma previously untreated or treated with chemotherapy (Patel, 2003) and (III) as first-line therapy in elderly patients with NSCLC of all histological subentities . The drug was well tolerated, drug-related cutaneous rash and diarrhea were observed in approximately two thirds of patients. The response rates were 12.3%, 25% and 13.3%, respectively. The median survival was 8.4 months, and the 1-year survival rate was 40% (Perez-Soler, 2004). A recently presented study with erlotinib at 150 mg as single agent in first line treatment of IIIB/IV NSCLC patients showed a non-progression rate of 55% after 6 weeks of treatment (Giaccone, 2005).
Based on the results of the single agent phase II studies, phase III studies in combination with chemotherapy were performed. In two large phase III studies (TALENT, TRIBUTE) analyzing treatment of erlotinib in combination with chemotherapy, in more than 2000 patients no benefit for the addition of erlotinib could be shown (Gatzemeier, 2004;Herbst, 2004).
Recently, the randomized, double blind, placebo controlled study performed by the National Cancer Institute of Canada evaluated 731 patients with locally advanced or metastatic (stage IIIb or IV) NSCLC after failure of one or more chemotherapy regimens. The proportion of patients who experienced an objective response in the erlotinib monotherapy group was higher than in the placebo group (8.9% versus 0.9%, p<0.001). Treatment with erlotinib was associated with significantly longer overall survival than placebo (median survival 6.7 months versus 4.7 months respectively) .
These data led to FDA approval in the US in November 2004. With the U.S label, Tarceva® (erlotinib) is indicated for all NSCLC patients after failure of at least one prior chemotherapy regimen. In 2005 Tarceva® has been approved in Switzerland and in the EU for the same indication.

Storage of Tarceva:
Tarceva will be provided by Roche and stored at room temperature in a temperature controlled, locked locker in the outpatient department of the department of internal medicine I. Unlabeled Tarceva® as commercially available will be provided by Roche.

Administration of therapy
Day 1-42 (week 1-6): Erlotinib (Tarceva®) 150 mg p.o. once daily, It is recommended to administer Tarceva® 1 hour after meals. In addition, patients should avoid food ingestion for at least 2 hours after Tarceva®. For the same reason the administration of proton pump blockers is not recommended during therapy.
It is recommended to perform the follwing blood tests routinely once per week during erlotinib (Tarceva®) therapy: EDTA blood: blood cell count serum: creatinin, uric acid, bilirubin, gamma-GT, GOT, GPT

Reported erlotinib (Tarceva®) adverse reactions
Clinically significant erlotinib (Tarceva®) adverse reactions: 1. Grade 3 to 4 rash and diarrhea occurred in 9% and 6% patients, respectively. The median time to onset of rash in a phase III clinical trial was 8 days and to onset of diarrhea 12 days .
2. Liver function abnormality -grade 2 (>2.5 -5.0 x ULN) including elevated ALT, AST and bilirubin occurred in 4% of the patients treated with Tarceva and <1% of placebo treated patients in a phase-III study. Grade 3 elevations were not observed in Tarceva® treated patients. During treatment with erlotinib, the patients should be monitored by liver function tests at least once every 1 to 2 months .
3. Interstitial pneumonia (interstitial lung disease -ILD) has been observed in a phase III study in NSCLC patients receiving erlotinib at an overall incidence of about 0.8 percent, which was not different to the placebo group. Among patients who developed ILD, symptoms began within 5 days to more than 9 months after erlotinib therapy initiation (median 47 days). The overall incidence of ILD in Tarceva® treated patients was reported to be approximately 0.6%. In the event of acute onset or worsening of pulmonary symptoms (dyspnea, cough, fever), erlotinib therapy should be interrupted and a prompt diagnostics of these symptoms should start. If interstitial lung disease is confirmed, erlotinib should be discontinued and the patient should be treated appropriately .
Adverse reactions occurring in Tarceva®-treated patients

NCI CTC Grade Any Grade (%) Grade 3 (%) Grade 4 (%)
Rash 75  4. If a patient develops diarrhoea, the patient should be managed appropriately by symptomatic treatment, dose reduction or temporary suspension of the treatment with erlotinib depending on the condition of the patient (see chapter 6.5 Treatment postponement, dose reductions and interruption).
5. In patients with elevated liver enzymes prior treatement (total bilirubin, ASAT, ALAT 2 x ULN) liver enzymes will be monitored twice weekly.
Precautions for co-administration with erlotinib (Tarceva®) Tarceva® should be administered with care when co-administered with following drugs: Drug Effects CYP3A4 inducers: phenytoin, carbamazepine, rifampicin, barbiturates, etc.
Blood erlotinib concentration may be decreased, which may lead to decreased effects of the drug.
Blood erlotinib concentrations may be increased, which may lead to increased incidence and severity of adverse drug reactions.
Co-administration with itraconazole resulted in an 80% increase in the AUC of erlotinib.

warfarin, cumarin (Marcumar)
There were reports on INR elevations and bleeding events on co-administration with warfarin. When erlotinib is used concomitantly with warfarin, the patient should be monitored for prothrombine time or INR at regular intervals.

Restaging procedures during and after erlotinib therapy Examinations
Since the primary objective of the proposed study is to evaluate the accuracy of FDG-/FLT-PET analysis for early prediction of erlotinib non-progression in patients with NSCLC, the FDG-and FLT-PET examinations will be performed in exactly defined periods before, during and after therapy (s. table below).

Therapeutic procedure after 6 weeks of erlotinib therapy
Only patients with documented (clinical examination, CT, PET) tumor control under erlotinib therapy (CR, PR or, at least, SD in the restaging procedures in week 7) will continue erlotinib therapy until progression of disease. In all patients with documented progress after 6 weeks of therapy erlotinib therapy will be stopped. Further treatment in these patients depends on overall performance state and declared intention of the patient. If the patient and the principal investigator or one of the co-investigators agree upon chemotherapeutic treatment, this treatment will be offered to the patient in the Department I of Internal Medicine, University Clinic Cologne.

Follow-up examinations
A standardized patient visit on month 3, 6, 9 and 12 comprises the following examinations: • CT scan of the involved area Reporting, Topic E2 will be adhered.
Any serious adverse event that is unequivocally due to disease progression should not be reported as an AE. It will be documented in the regular CRF. Laboratory abnormalities of at least grade 2 NCI-CTCAE v3.0 will be documented as AEs.
The trial coordinating center will forward any clinical adverse event or abnormal laboratory test value that is serious to Roche within one working day. Roche will fulfill reporting requirements to authorities and ethics committees. SUSARs will be distributed to ethics committees and centers by Roche.

Treatment and Follow-up of Adverse Events
Adverse events, especially those for which the relationship to test "drug" is not "unrelated", should be followed up until they have returned to baseline status or stabilized. If a clear explanation is established it should be recorded on the CRF.

Follow-up of Abnormal Laboratory Test Values
In the event of unexplained abnormal laboratory test values, the tests should be repeated immediately and followed up until they have returned to the normal range and/or an adequate explanation of the abnormality is found. If a clear explanation is established it should be recorded on the CRF.

Pregnancy
A female subject must be instructed to stop taking the test "drug" and immediately inform the investigator if she becomes pregnant during the study. Pregnancies occurring up to 90 days after the completion of the test "drug" must also be reported to the investigator. The investigator must report all pregnancies within one working day to the sponsor. The investigator should counsel the subject, discuss the risks of continuing with the pregnancy and the possible effects on the fetus. Monitoring of the patient should continue until conclusion of the pregnancy.
Pregnancy occurring in the partner of a subject participating in the study should also be reported to the investigator and the sponsor. The partner should be counseled and followed as described above.

Definitions
An adverse event is any untoward medical occurrence in a patient or clinical investigation subject, administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment.
Adverse reactions are all untoward and unintended responses to an investigational medicinal product related to any dose administered.
A serious adverse event or serious adverse reaction is any experience that suggests a significant hazard, contraindication, side effect or precaution. It is any adverse event that at any dose fulfills at least one of the following criteria: • is fatal (results in death) (NOTE: death is an outcome, not an event) • is life-threatening (NOTE: the term "life-threatening" refers to an event in which the patient was at immediate risk of death at the time of the event; it does not refer to an event which could hypothetically have caused a death had it been more severe.) • required in-patient hospitalization or prolongation of existing hospitalization • results in persistent or significant disability/incapacity • is a congenital anomaly/birth defect • is medically significant or requires intervention to prevent one or other of the outcomes listed above Medical and scientific judgment should be exercised in deciding whether expedited reporting to the sponsor is appropriate in other situations, such as important medical events that may not be immediately life-threatening or result in death or hospitalization but may jeopardize the patient or may require intervention to prevent one of the outcomes listed in the definitions above. These situations should also usually be considered serious.
Examples of such events are intensive treatment in an emergency room or at home for allergic bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; or development of drug dependency or drug abuse.
An unexpected adverse event is one, the nature or severity of which is not consistent with the applicable product information.
Causality is initially assessed by the investigator. With respect to report and documentation obligation (regulatory authorities, ethics committees and other investigators) for Serious Adverse Events, causality can be one of 2 possibilities: • No (unrelated; equals not drug related).
All adverse events not assessed as definitive "not drug related" by either the investigator or Roche will be considered as adverse drug reaction.
A suspected unexpected serious adverse reaction (SUSAR) is a serious adverse reaction, the nature, or severity of which is not consistent with the applicable product information.
It is important that the severity of an adverse event is not confounded with the seriousness of the event. For example, vomiting which persists for many hours may be severe, but is not necessarily a serious adverse event. On the other hand, stroke which results in only a limited degree of disability may be considered a mild stroke, but would be a serious adverse event.
A serious adverse event occurring during the study or which comes to the attention of the investigator within 28 days after stopping the treatment, whether considered treatmentrelated or not, must be reported. In addition, a serious adverse event that occurs after this time, if considered related to test "drug", should be reported.
Such preliminary reports will be followed by detailed descriptions later which will include copies of hospital case reports, autopsy reports and other documents.
For serious adverse events, the following must be assessed and recorded on the adverse events page of the Case Report Form: intensity, relationship to test substance, action taken, and outcome to date.
Document and report obligation have to be adhered according to the national and international laws and regulations.

Treatment postponement, dose reduction and termination.
In case of any adverse event, which may be classified by the investigators as an suspected drug reaction and is not controlled by optimal supportive care, or not tolerated due to symptoms, disfigurement, or interference with normal daily activities, Tarceva® dose may be reduced according to the following schedule: Starting dose: 150 mg First reduction: 100 mg Second reduction: 50 mg.
Within further 2 weeks after dose reduction, the adverse event must have been improved at least by one NCI-CTC grade to NCI-CTC grade < 2. Otherwise, a further dose reduction must be performed. The dose interruption may last maximal 2 weeks if clinically indicated and if the toxicity is not controlled by optimal therapy.
Once a dose was reduced due to toxicity, the dose will not be reescalated, except for Tarceva® induced rash. In case of rash, the dose can be reescalated if the rash is < grade 2 NCI-CTC.
In case of skin reaction and diarrhoea the following dose management and therapy is indicated: off study

NCI -CTC Grade
Patients requiring a dose interruption for > 2 weeks due to not controlled adverse events of the Tarceva® medication will be taken off study.
In any case of a grade IV toxicity the patient will be withdrawn from the study. Any grade III toxicity will be reported to the principal investigator within 24 h after notice.

Handling of FDG and FLT safety
No adverse events were described after administration of FLT and FDG up to now. In any case of adverse event, especially allergic reaction, the next dose of FLT or FDG, respectively, will not be administered.

Statistical formulation of study objectives and design Primary Objective
To evaluate the accuracy of FDG-/FLT-PET analyses for early prediction of nonprogression in patients with NSCLC treated with erlotinib At least a 30% decrease in the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD * Progressive Disease (PD): At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions * Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started * Non-progression: CR + PR + SD but not PD Hypotheses, clinical 1. A difference in early FDG-or FLT-PET examinations between progressers (PD) and nonprogressers (CR, PR, SD) in NSCLC patients treated with erlotinib can be detected.
2. EGFR mutations are found in patients with CR or PR and KRAS mutations in patients with PD after treatment with erlotinib.

Trial design
The trial will be performed as a single-arm, single-center, open-label study.

Recruitment
Patients will be recruited after screening only if all inclusion criteria are met, no exclusion criteria are met and written informed consent has been obtained.

Cancellation of recruitment
The recruitment of patients who are not qualified for the trial can be cancelled. Evidence that the patient is not qualified must be based on data obtained before inclusion (e.g., histology, staging, case history or previous treatment). Only for those patients who were recruited in error without knowledge of these data recruitment can be cancelled. Those for whom a physician later assesses the findings differently or discovers additional involved sites over the course of time (e.g. because of delays in treatment) cannot be cancelled.

Interim analyses
No interim analysis is planned.

Early stopping strategy
The trial will be stopped early if unexpectedly high rates of SAEs are observed.

Analyses of primary endpoints
1. The prognostic value/accuracy of FDG-/FLT-PET regarding non-progression in NSCLC patients treated with erlotinib will be evaluated by ROC analysis (area under the ROC curve, AUC); H 0 : AUC FDG/FLT PET ≤ 0.5, H A : AUC FDG/FLT PET > 0.5 (intersection-union test at one-sided level 2.5% -primary objective).
2. The statistical association of EGFR respectively KRAS mutational status, EGFR positivity and clinical response in NSCLC patients treated with erlotinib will be evaluated by Fisher's exact test (at one-sided level 2.5%; strong type 1 error control is maintained if analysis of item 1 was significant -secondary objective).

Analysis of secondary endpoints
The prognostic value/accuracy of FDG/FLT-PET regarding clinical failure, SD and clinical response in NSCLC patients treated with erlotinib will be compared by ROC analysis; optimal cut-off values for FDG/FLT PET will be chosen (secondary objective).
Association between EGFR mutation, KRAS mutation and EGFR positivity with clinical response will be tested by Fisher´s exact test.
Secondary endpoints (FFTF and OS) will be analyzed using descriptive statistics after at least one year follow-up of all patients.

Statistical Reporting
The final statistical report will be done by PD Dr. M. Hellmich and included also in the final report of the study.

Expected results
Based on the results of clinical trials with erlotinib in NSCLC patients in Western Europe and the US, we expect about 10% of patients with response (CR, PR) to treatment with erlotinib, about 30% with stable disease and about 60% with progressive disease. Furthermore, we expect about 90% of patients with mutated EGFR responding to therapy. KRAS mutations and EGFR mutations are expected to be nearly exclusive.
Based on published PET data, especially from patients with GIST treated with imatinib, we expect a nearly 100% association of clinical response (CR, PR in CT after 6 weeks of treatment) and reduction in SUV (PET analyses on days 8,9).

Expected flow of information
For the expected patient population similar response rates as described in published studies for Western World patients are expected.

Subgroup analysis
To evaluate differential results concerning the predictive value of the PET analysis between male and female patients a separate analysis for both groups will be performed.

Data analysis
Raw data of PET will be analyzed by two experienced nuclear physicians in consensus by visual control and semiquantitative (Standardized Uptake Value, SUV) methods using the MPI-tool with knowledge of the CT-findings. Results of the diagnostic procedures will be documented in the CRF on the same day or up to 3 days thereafter latest. Tomograms will be analyzed in all three planes (coronar, sagital, transversal). Data will be correlated with computed tomography. Data will be stored for direct comparison in the follow-up.
Raw data of CT will be analyzed by two experienced radiologists in consensus according to the RECIST criteria (Therasse et al., 2000) with knowledge of the FDG-PET and FLT-PET.
Results of the diagnostic procedures will be documented in the CRF on the same day or up to 3 days thereafter latest.
To assure data quality, an external review of imaging findings will be performed after enrollment of the last patient.
Statistical analysis will be performed by M. Hellmich. The ROC analysis will be performed nonparametrically according to (DeLong et al., 1988).

Inclusion in the analysis
Full analysis set (primary analysis, intention-to-treat): All patients included in the study will be analysed (contingent on availability of endpoints). Per-protocol set (secondary analysis): Only patients fully compliant with the protocol (especially regarding dosage) will be analysed.
The full analysis set will be taken as safety analysis population as all patients will receive the study medication.

DOCUMENTATION AND MONITORING
All data collected are documented on standardized case report forms (CRF) by the treating physician or other persons given the responsibility for documentation.
A multilevel plan for data validation has been drawn up. In case of missing or inconsistent data, a data manager will contact the treating physician or person responsible for documentation in the department by telephone or in writing (queries).

Direct access to source data
A regulatory authority may wish to conduct an inspection (during the trial or even after its completion). If an inspection is requested by a regulatory authority, the investigator must inform the trial coordination center immediately that this request has been made. The investigator will permit trial-related monitoring, audits, reviews by the independent ethics committee, and regulatory inspections, providing direct access to source data and documents.

Quality control and assurance
Each investigator is responsible at his site that the trial is performed in accordance with GCP, AMG and the protocol. He ensures that data is collected as stated in the protocol and documented correctly in the responding CRF. Monitors will assist investigators in reviewing the complete, legible, clearly arranged and recallable data. All data is examined by the monitors.

Monitoring
In order to guarantee a high quality of the trial and data retrieval, all participating centers will be visited on a regular base on site by monitors.
Data protection rights will be respected. Patient files will be analyzed on a random base to control original data and to verify accurate data registration and management. The presence of a written informed consent form and the correct interpretation of inclusion and exclusion criteria will be controlled. The monitor will also have regular contact by phone and/or e-mail with all participating centers.
The investigator agrees that the trial center will be visited on a regular base on site by the monitor and will support the monitor in his/her activities.
Purpose of this visits are: • Evaluation of the trial progression • Adherence to the trial protocol • Discussion of problems related to trial problems (adverse events) • Check of the CRFs concerning accuracy and completeness • Validation of the CRFs compared to the source data • Control of the administration of the trial medication The monitor writes a monitoring-report about each visit. In this monitoring report will be documented the trial progress and all appearing problems.

Documentation
All protocol required procedures along with information necessary to report the observations and tests described in this protocol will be recorded in the Case report forms (CRFs). CRFs must be completed and given to the trial coordination center in a timely and accurate manner by site personnel.
CT scans must be reported in summary in the CRF. The original reports, traces and films must be retained by the investigator for future reference.
The investigator must review all pages within the CRF for accuracy and consistency with the protocol, and sign and date the CRF sign-off page(s) upon completion.
Corrections to data on the CRFs must be made by lining out the incorrect data with a single line and writing the correct data near to those crossed out. Correction fluid is not to be used to cover errors. Each correction must be initialed and dated by the person making the correction.
The patient consent forms designated for the clinical investigator are also to be kept in the trial documentation. All the information on which the entries in the CRF are based must be available in the patient files e. g. results of laboratory investigations.
After prior agreement, a check of the consistency of data between the patient files, raw data and CRF as well as with other documents related to the trial will be conducted by the responsible authorities and/or by monitors (inspection/audit/monitoring).
The clinical investigator is responsible for the identity of the data in the patient file and the correct entry of the data into the CRF.

Data management
All incoming CRF and DCF will be registered by the data management. All registration forms will be checked for completeness, plausibility, and correctness. Missing, questionable, or additional information will be requested on a regular base by the data management. The data management will remind for missing documentation regularly.
Data will be entered into an Oracle database using MACRO as the data entry application.
During entry data will be checked by constraints and triggers.
The database will be checked for faults and validated by the database programmer.
Thereafter the database will approve the database for data entry. Complete and incremental data backup will be performed regularly.
All CRFs and other essential trial documents will be archived for at least 15 years. Patients ID-lists and medical records will be kept separately at the individual trial sites.

Record keeping
A copy of the final completed CRFs is retained by the investigator, who must ensure that it is stored with other trial documents, such as the protocol, the investigator's brochure and any protocol amendments, in a secure place.
Case records and raw data are to be retained for at least 15 years following the end of the trial. The investigator will ensure that a correct assignment of the CRFs to the corresponding patient files and raw data is possible at any time. A separate patient identification list must be recorded by the investigator. Patients ID-lists and medical records will be kept separately at the individual trial sites by the investigator.

ETHICAL MATTERS
The trial is performed according to the declaration of Helsinki (Helsinki, 2000) and the principles of the ICH guidelines on Good Clinical Practice (Spira and Ettinger, 2004). The ICH guidelines have been adapted to the conditions applying to quality assurance protocols in clinical trials groups.
The trial coordinators have obtained a vote of approval from the local responsible ethical committee -(Ethikkommission der Universität zu Köln) of the responsible authorities (BfArM and BFS) according to the German drug regulations (Arzneimittelgesetzt, 2004).
For PET diagnostics, the trial coordinators will obtain the approval of the German Authority for Radiation Safety (Bundesamt für Strahlenschutz). As synthesis and application of FLT and FDG is performed within the trial center, §4a.3 of the AMG is applicable.
Other radiation burdens (radiotherapy, X-rays) lie within the range of usual medical care and approval is therefore, under present legislation, not required.
The trial coordinators will obtain approvement the responsible ethical committee, according to the German drug regulations, whenever the trial protocol is amended such as to require renewed assessment of risk to patients (e.g. changes to dose or to duration of treatment, or the occurrence of serious adverse events).
Roche will fulfill reporting requirements to authorities and ethics committees. SUSARs will be distributed to ethics committees and centers by Roche.

Retrospective protocol amendments
Protocol amendments or additions can be agreed upon by the trial coordinators on the basis of current information in the literature for reasons of ethics or safety.
Any protocol amendment is to be: • Approved by the ethical committee and the regulatory authority if the risk to patients must be newly assessed; • recorded in the protocol logbook, with date

Insurance
Insurance (Gerling) is provided for the patients in this trial. Details can be obtained from the trial coordination center.

Agreement on publication
Analyses and publications (lectures, articles, etc.) of data and information connected with the present quality assurance protocol may be made solely with the agreement of the LCGC trial coordinator. Any formal presentation or publication of data collected from this trial will be considered as a joint publication by the investigators, the principal investigators, and the sponsor. Authorship will be determined by mutual agreement.  2. It is the duty of the physician to promote and safeguard the health of the people. The physician's knowledge and conscience are dedicated to the fulfillment of this duty.
3. The Declaration of Geneva of the World Medical Association binds the physician with the words, "The health of my patient will be my first consideration," and the International Code of Medical Ethics declares that, "A physician shall act only in the patient's interest when providing medical care which might have the effect of weakening the physical and mental condition of the patient." 4. Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects.
5. In medical research on human subjects, considerations related to the well-being of the human subject should take precedence over the interests of science and society.
6. The primary purpose of medical research involving human subjects is to improve prophylactic, diagnostic and therapeutic procedures and the understanding of the aetiology and pathogenesis of disease. Even the best proven prophylactic, diagnostic, and therapeutic methods must continuously be challenged through research for their effectiveness, efficiency, accessibility and quality.
7. In current medical practice and in medical research, most prophylactic, diagnostic and therapeutic procedures involve risks and burdens.
8. Medical research is subject to ethical standards that promote respect for all human beings and protect their health and rights. Some research populations are vulnerable and need special protection. The particular needs of the economically and medically disadvantaged must be recognized. Special attention is also required for those who cannot give or refuse consent for themselves, for those who may be subject to giving consent under duress, for those who will not benefit personally from the research and for those for whom the research is combined with care.
9. Research Investigators should be aware of the ethical, legal and regulatory requirements for research on human subjects in their own countries as well as applicable international requirements. No national ethical, legal or regulatory requirement should be allowed to reduce or eliminate any of the protections for human subjects set forth in this Declaration.

B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH
10. It is the duty of the physician in medical research to protect the life, health, privacy, and dignity of the human subject.
11. Medical research involving human subjects must conform to generally accepted scientific principles, be based on a thorough knowledge of the scientific literature, other relevant sources of information, and on adequate laboratory and, where appropriate, animal experimentation.
12. Appropriate caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected.
13. The design and performance of each experimental procedure involving human subjects should be clearly formulated in an experimental protocol. This protocol should be submitted for consideration, comment, guidance, and where appropriate, approval to a specially appointed ethical review committee, which must be independent of the investigator, the sponsor or any other kind of undue influence. This independent committee should be in conformity with the laws and regulations of the country in which the research experiment is performed. The committee has the right to monitor ongoing trials. The researcher has the obligation to provide monitoring information to the committee, especially any serious adverse events. The researcher should also submit to the committee, for review, information regarding funding, sponsors, institutional affiliations, other potential conflicts of interest and incentives for subjects.
14. The research protocol should always contain a statement of the ethical considerations involved and should indicate that there is compliance with the principles enunciated in this Declaration.
15. Medical research involving human subjects should be conducted only by scientifically qualified persons and under the supervision of a clinically competent medical person. The responsibility for the human subject must always rest with a medically qualified person and never rest on the subject of the research, even though the subject has given consent.
16. Every medical research project involving human subjects should be preceded by careful assessment of predictable risks and burdens in comparison with foreseeable benefits to the subject or to others. This does not preclude the participation of healthy volunteers in medical research. The design of all studies should be publicly available.
17. Physicians should abstain from engaging in research projects involving human subjects unless they are confident that the risks involved have been adequately assessed and can be satisfactorily managed. Physicians should cease any investigation if the risks are found to outweigh the potential benefits or if there is conclusive proof of positive and beneficial results.
18. Medical research involving human subjects should only be conducted if the importance of the objective outweighs the inherent risks and burdens to the subject. This is especially important when the human subjects are healthy volunteers.
19. Medical research is only justified if there is a reasonable likelihood that the populations in which the research is carried out stand to benefit from the results of the research.
20. The subjects must be volunteers and informed participants in the research project.
21. The right of research subjects to safeguard their integrity must always be respected. Every precaution should be taken to respect the privacy of the subject, the confidentiality of the patient's information and to minimize the impact of the study on the subject's physical and mental integrity and on the personality of the subject.
22. In any research on human beings, each potential subject must be adequately informed of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliations of the researcher, the anticipated benefits and potential risks of the study and the discomfort it may entail. The subject should be informed of the right to abstain from participation in the study or to withdraw consent to participate at any time without reprisal. After ensuring that the subject has understood the information, the physician should then obtain the subject's freely-given informed consent, preferably in writing. If the consent cannot be obtained in writing, the non-written consent must be formally documented and witnessed.
23. When obtaining informed consent for the research project the physician should be particularly cautious if the subject is in a dependent relationship with the physician or may consent under duress. In that case the informed consent should be obtained by a well-informed physician who is not engaged in the investigation and who is completely independent of this relationship.
24. For a research subject who is legally incompetent, physically or mentally incapable of giving consent or is a legally incompetent minor, the investigator must obtain informed consent from the legally authorized representative in accordance with applicable law. These groups should not be included in research unless the research is necessary to promote the health of the population represented and this research cannot instead be performed on legally competent persons.
25. When a subject deemed legally incompetent, such as a minor child, is able to give assent to decisions about participation in research, the investigator must obtain that assent in addition to the consent of the legally authorized representative.
26. Research on individuals from whom it is not possible to obtain consent, including proxy or advance consent, should be done only if the physical/mental condition that prevents obtaining informed consent is a necessary characteristic of the research population. The specific reasons for involving research subjects with a condition that renders them unable to give informed consent should be stated in the experimental protocol for consideration and approval of the review committee. The protocol should state that consent to remain in the research should be obtained as soon as possible from the individual or a legally authorized surrogate.

C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH MEDICAL CARE
28. The physician may combine medical research with medical care, only to the extent that the research is justified by its potential prophylactic, diagnostic or therapeutic value. When medical research is combined with medical care, additional standards apply to protect the patients who are research subjects.
29. The benefits, risks, burdens and effectiveness of a new method should be tested against those of the best current prophylactic, diagnostic, and therapeutic methods. This does not exclude the use of placebo, or no treatment, in studies where no proven prophylactic, diagnostic or therapeutic method exists. See footnote 30. At the conclusion of the study, every patient entered into the study should be assured of access to the best proven prophylactic, diagnostic and therapeutic methods identified by the study.
31.The physician should fully inform the patient which aspects of the care are related to the research. The refusal of a patient to participate in a study must never interfere with the patient-physician relationship.
32. In the treatment of a patient, where proven prophylactic, diagnostic and therapeutic methods do not exist or have been ineffective, the physician, with informed consent from the patient, must be free to use unproven or new prophylactic, diagnostic and therapeutic measures, if in the physician's judgement it offers hope of saving life, re-establishing health or alleviating suffering. Where possible, these measures should be made the object of research, designed to evaluate their safety and efficacy. In all cases, new information should be recorded and, where appropriate, published. The other relevant guidelines of this Declaration should be followed.

FOOTNOTE: NOTE OF CLARIFICATION ON PARAGRAPH 29 of the WMA DECLARATION OF HELSINKI
The WMA hereby reaffirms its position that extreme care must be taken in making use of a placebocontrolled trial and that in general this methodology should only be used in the absence of existing proven therapy. However, a placebo-controlled trial may be ethically acceptable, even if proven therapy is available, under the following circumstances: -Where for compelling and scientifically sound methodological reasons its use is necessary to determine the efficacy or safety of a prophylactic, diagnostic or therapeutic method; or -Where a prophylactic, diagnostic or therapeutic method is being investigated for a minor condition and the patients who receive placebo will not be subject to any additional risk of serious or irreversible harm.
All other provisions of the Declaration of Helsinki must be adhered to, especially the need for appropriate ethical and scientific review.

Vote of approval by the ethical committee of Universitätsklinikum Köln (original)
The ERLOPET clinical study protocol is currently submitted to the ethics committee of the University Hospital of Cologne.

Activity index according to the ECOG
The ECOG activity index assesses the performance status of the patient with regard to physical and social factors: 0 Fully active, able to carry on all pre-disease activities without restrictions.
1 Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature e.g. light house work, office work.
2 Ambulatory and capable of all self care but unable to carry out any work activities. Up and about more than 50% of waking hours.

3
Capable of only limited self care, confined to bed or chair more than 50% of waking hours.

4
Completely disabled. Cannot carry on any self care. Totally confined to bed or chair.

Response Evaluation Criteria in Solid Tumors (RECIST) Quick Reference Eligibility (http://www3.cancer.gov/bip/RECIST.htm)
• Only patients with measurable disease at baseline should be included in protocols where objective tumor response is the primary endpoint.
Measurable disease -the presence of at least one measurable lesion. If the measurable disease is restricted to a solitary lesion, its neoplastic nature should be confirmed by cytology/histology.
Measurable lesions -lesions that can be accurately measured in at least one dimension with longest diameter ≥20 mm using conventional techniques or ≥10 mm with spiral CT scan.
Non-measurable lesions -all other lesions, including small lesions (longest diameter <20 mm with conventional techniques or <10 mm with spiral CT scan), i.e., bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusion, inflammatory breast disease, lymphangitis cutis/pulmonis, cystic lesions, and also abdominal masses that are not confirmed and followed by imaging techniques; and.
• All measurements should be taken and recorded in metric notation, using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment.
• The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up.
• Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes). For the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.

Methods of Measurement
• CT and MRI are the best currently available and reproducible methods to measure target lesions selected for response assessment. Conventional CT and MRI should be performed with cuts of 10 mm or less in slice thickness contiguously. Spiral CT should be performed using a 5 mm contiguous reconstruction algorithm. This applies to tumors of the chest, abdomen and pelvis. Head and neck tumors and those of extremities usually require specific protocols.
• Lesions on chest X-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable.
• When the primary endpoint of the study is objective response evaluation, ultrasound (US) should not be used to measure tumor lesions. It is, however, a possible alternative to clinical measurements of superficial palpable lymph nodes, subcutaneous lesions and thyroid nodules. US might also be useful to confirm the complete disappearance of superficial lesions usually assessed by clinical examination.
• The utilization of endoscopy and laparoscopy for objective tumor evaluation has not yet been fully and widely validated. Their uses in this specific context require sophisticated equipment and a high level of expertise that may only be available in some centers. Therefore, the utilization of such techniques for objective tumor response should be restricted to validation purposes in specialized centers. However, such techniques can be useful in confirming complete pathological response when biopsies are obtained.
• Tumor markers alone cannot be used to assess response. If markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response when all lesions have disappeared.
• Cytology and histology can be used to differentiate between PR and CR in rare cases (e.g., after treatment to differentiate between residual benign lesions and residual malignant lesions in tumor types such as germ cell tumors).

Baseline documentation of "Target" and "Non-Target" lesions
• All measurable lesions up to a maximum of five lesions per organ and 10 lesions in total, representative of all involved organs should be identified as target lesions and recorded and measured at baseline.
• Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repeated measurements (either by imaging techniques or clinically).
• A sum of the longest diameter (LD) for all target lesions will be calculated and reported as the baseline sum LD. The baseline sum LD will be used as reference by which to characterize the objective tumor.
All other lesions (or sites of disease) should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but the presence or absence of each should be noted throughout followup.

Evaluation of target lesions
Complete Response ( (1) (1) Although a clear progression of "non target" lesions only is exceptional, in such circumstances, the opinion of the treating physician should prevail and the progression status should be confirmed later on by the review panel (or study chair).

Evaluation of best overall response
The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for PD the smallest measurements recorded since the treatment started). In general, the patient's best response assignment will depend on the achievement of both measurement and confirmation criteria • Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having "symptomatic deterioration". Every effort should be made to document the objective progression even after discontinuation of treatment.
• In some circumstances it may be difficult to distinguish residual disease from normal tissue. When the evaluation of complete response depends on this determination, it is recommended that the residual lesion be investigated (fine needle aspirate/biopsy) to confirm the complete response status.

Confirmation
• The main goal of confirmation of objective response is to avoid overestimating the response rate observed. In cases where confirmation of response is not feasible, it should be made clear when reporting the outcome of such studies that the responses are not confirmed.
• To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by repeat assessments that should be performed no less than 4 weeks after the criteria for response are first met. Longer intervals as determined by the study protocol may also be appropriate.
• In the case of SD, follow-up measurements must have met the SD criteria at least once after study entry at a minimum interval (in general, not less than 6 weeks) that is defined in the study protocol

Duration of overall response
• The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever status is recorded first) until the first date that recurrence or PD is objectively documented, taking as reference for PD the smallest measurements recorded since the treatment started.

Duration of stable disease
• SD is measured from the start of the treatment until the criteria for disease progression are met, taking as reference the smallest measurements recorded since the treatment started.
• The clinical relevance of the duration of SD varies for different tumor types and grades. Therefore, it is highly recommended that the protocol specify the minimal time interval required between two measurements for determination of SD. This time interval should take into account the expected clinical benefit that such a status may bring to the population under study.

Response review
• For trials where the response rate is the primary endpoint it is strongly recommended that all responses be reviewed by an expert(s) independent of the study at the study's completion. Simultaneous review of the patients' files and radiological images is the best approach.

Reporting of results
• All patients included in the study must be assessed for response to treatment, even if there are major protocol treatment deviations or if they are ineligible. Each patient will be assigned one of the following categories: 1) complete response, 2) partial response, 3) stable disease, 4) progressive disease, 5) early death from malignant disease, 6) early death from toxicity, 7) early death because of other cause, or 9) unknown (not assessable, insufficient data).
• All of the patients who met the eligibility criteria should be included in the main analysis of the response rate. Patients in response categories 4-9 should be considered as failing to respond to treatment (disease progression). Thus, an incorrect treatment schedule or drug administration does not result in exclusion from the analysis of the response rate. Precise definitions for categories 4-9 will be protocol specific.
• All conclusions should be based on all eligible patients.
• Subanalyses may then be performed on the basis of a subset of patients, excluding those for whom major protocol deviations have been identified (e.g., early death due to other reasons, early discontinuation of treatment, major protocol violations, etc.). However, these subanalyses may not serve as the basis for drawing conclusions concerning treatment efficacy, and the reasons for excluding patients from the analysis should be clearly reported.
• The 95% confidence intervals should be provided.

Patient preparation
For FDG-PET and FLT-PET patients must refrain from ingestion for 12 hours prior to the PET examination; only the ingestion of carbohydrate-free drinks is permitted. The current glucose level should be measured before starting FDG-PET. Fasting glucose level over 130 mg/dl or known diabetes mellitus are exclusion criteria for FDG-PET. The physicians may optionally administer regular insulin (e.g. 2 units) if blood glucose levels are unexpectedly high, provided that monitoring and emergency care are assured. For FLT-PET no special preparation is necessary.

Administration of tracers, scanning and data evaluation
18F-FDG and 18F-FLT will be synthesized at the Max-Planck-Institute for Neurological Research at the University of Cologne following standard procedures. A dose of 370 MBq FDG and 300 MBq FLT will be injected intravenously for whole-body examination by PET. Scanning will commence ~45-60 minutes after injection of tracers. PET instruments with fullring detectors will be applied. Emission data will be corrected for attenuation, scatter and random and will be reconstructed to plains of 3.125 mm width and 128x128 voxels. For quantification of tracer uptake, standard methods, such as standardized uptake values (SUV) will be applied.

Sample procurement
Samples will be obtained from the same tumor biopsy specimen used for histological diagnosis. The primary pathologist decides which part of a sample can be used for biological analyses. All sample procurement and storage processes have already been established at MTBTI, Dept I of Internal Medicine, University Clinic Cologne and are highly standardized as standard operating procedures (SOPs).

Estimation of tumor cell purity and preparation of genomic DNA
The primary pathologist will estimate the tumor cell purity at the margin of dissection where the snap-frozen sample has been cut off. When the tumor cell population is estimated to be >70%, DNA will be directly prepared from 20 µm sections prepared using standard section techniques. DNA will be prepared using the Puregene™ kit (Gentra), following the recommendations of the manufacturer. If the tumor purity is estimated to be <70%, tumor cells will be enriched using a laser-pressure catapulting microdissection instrument (PALM Microlaser Technologies). DNA will be purified from these samples using the Puregene™ kit.

Amplification and direct sequencing of the EGFR and KRAS
Pre-treatment biopsy material will be analyzed for EGFR mutational status by exon resequencing. Exon-spanning primers will be designed using Primer3 software in the introns adjacent to exons and target sequences will be amplified by single-round PCR. Purified PCR products will be bi-directionally sequenced (sequences controlled for bi-allelic target amplification; quality threshold: Phred score >30).

Sequence analyses
The resulting electropherograms will be manually and computationally (software: DNAsis) compared to germline sequences of the EGFR gene and KRAS gene (GeneID: 1956; RefSeq: NM_005228, GeneID 3845, RefSeq: NM_033360). Splice sites will also be analyzed.
Sequence variations will be classified mutations if they are present in both bi-directional target electrophergrams and if they do not correspond to known germline polymorphisms. Furthermore, sequencing of control DNA from non-tumoral tissue (peripheral blood) is mandatory in those cases with a mutation to control for rare germline polymorphisms.

Quality control
Quality control is according to the recommendations for tumor depiction with FDG of the German Society for Nuclear Medicine (Knapp 1999).

Procedure for the performance of computed tomography
To optimize the assessment of tumor tissue volume using computed tomography, the following technical criteria should be aimed at: • Spiral CT technique • Intravenous contrast medium administered for all examinations • Oral contrast medium for abdominal CT • Spiral CT neck: (collimation/table shift/increment) 5/6/4 • Spiral CT chest: 5/8/4 • Spiral CT abdomen: 8/10/8 Computed tomography is inadequate: • with layer thickness > 1 cm and table shift > 1.5 cm • without or with too weak intravenous contrasting, which fails to differentiate blood vessels from neighboring tumor and tumor-affected lymphatic tissue reliably • without or with too weak oral contrasting, which fails to differentiate intestine from neighboring lymphoma tissue reliably.

Guide to patient briefing
Patient briefing is to contain all the points 1. to 6. below. A witness is required only if the patient is illiterate.

The trial
Short explanation of the trial title, verdict of the ethical committee, recruitment, insurance, aim, experimental procedure, procedure and advantages of the trial, transfer of data and test material, freedom of decision, absence of additional trial-related costs.

Nature of the disease
Short description of symptoms, clinical matters, response to treatment and prognosis as well as age distribution and the prognosis of untreated disease.

A.
Target group: NSCLC patients in stage IIIB/IV B.

Treatment:
Therapy with erlotinib (Tarceva®) until week 6 In the course of the study patients must undergo repeated PET examinations to evaluate response to therapy.

Aim:
To evaluate the accuracy of FDG-/FLT-PET analyses for early prediction of erlotinib non-progression in patients with NSCLC D.

Effects:
Lessening of symptoms such as weight loss, loss of appetite, cough, hemoptysis, dyspnea, chronical pneumonia; improvement of general wellbeing.

Possible side effects:
Mainly mild diarrhea and rashes were observed. In 0.6% of the patients interstitial lung disease was observed.

Approval:
Erlotinib is approved in the EU (including Germany), Switzerland and the US for the treatment of non-small cell lung cancer after failure of at least one prior chemotherapy regimen. Erlotinib so far is not approved for first line treatment of lung cancer. In phase II trials, however, erlotinib has shown therapeutic activity in patients with NSCLC treated in the first line situation comparable to chemotherapy. Tarceva is the only drug in the EGFR inhibitor class to demonstrate an increase in overall survival in a phase III clinical trial in advanced NSCLC patients. There is thus experience with the safety issues connected with this drug. Importantly, the adverse events reported for erlotinib are mild as compared to those of chemotherapy. Moreover, erlotinib has been reported to be at least as effective as chemotherapy in the treatment of relapsed lung cancer.

G.
PET: PET will be performed for identification of an early response to treatment with erlotinib. Both tracers (FDG and FLT) are well tolerated, allergic reactions are only very rarely observed. FLT is not approved for PET analyses in lung cancer. However, FLT has been successfully administered to more than 150 patients worldwide, up to now no adverse event has been reported. Moreover, at the Max-Planck Institute for Neurological Research at the University of Cologne more than 40 patients have been investigated using FLT-PET and no single adverse event has been noted, up to now. Additionally, this indicates an extensive experience with the synthesis and administration of FLT in humans. Beyond the radioactivity issues connected with FLT, this tracer can thus be considered safe.

Follow-up examinations
It is explained to the patient that regular monitoring examinations over a time-span till the follow up period end date are required in his/her own interest and especially in the interest of future patients. The results of these examinations will be reported to the trials coordination center.
The patient is told that he/she can be contacted by the trials coordination center, if he/she has agreed thereto.

Patients freedom of decision
The patient can reconsider his decision to participate in the trial at any time. If the patient refuses participation, he/she is free to choose further treatment.

Further procedures before patient´s consent.
After the patient´s information the patient will be asked for his/her queries and he/she will have at least 24 h in addition to consider his/her participation in the trial. The written informed consent of the patient has to be obtained before any study-related activities are carried out. It must be signed and personally dated by the patient and by the investigator/person designated by the investigator to conduct the informed consent discussion. To avoid any misunderstandings, the informed consent should be signed by the patient in the presence of the investigator/person designated by the investigator to conduct the informed consent discussion. The signed and dated declarations of informed consent will remain at the investigator´s site and must be safely archived by the investigator so that the forms can be retrieved at any time for monitoring, auditing and inspection purposes. A copy of the signed and dated information and consent should be provided to the patient prior to participation. Nach Abschluss der vollständigen diagnostischen Untersuchungen, die etwa eine Woche beanspruchen, kann zügig mit der Therapie begonnen werden.