Gemcitabine Combined with Gefitinib in Patients with Inoperable or ...

Cancer Investigation, 26:784–793, 2008 ISSN: 0735-7907 print / 1532-4192 online c Informa Healthcare USA, Inc. Copyright DOI: 10.1080/07357900801918611

ORIGINAL ARTICLE Clinical Translational Therapeutics

Gemcitabine Combined with Gefitinib in Patients with Inoperable or Metastatic Pancreatic Cancer: A Phase II Study of the Hellenic Cooperative Oncology Group with Biomarker Evaluation George Fountzilas,1 Mattheos Bobos,1 Anna Kalogera-Fountzila,1 Nikolaos Xiros,2 Samuel Murray,3 Helena Linardou,3 Georgia Karayannopoulou,1 Angelos K. Koutras,4 Dimitrios Bafaloukos,3 Epaminondas Samantas,5 Christos Christodoulou,6 Theofanis Economopoulos,2 Konstantine T. Kalogeras,1,7 and Paris Kosmidis8 Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece1 University General Hospital “Attikon”, Athens, Greece2 “Metropolitan” Hospital, Piraeus, Greece3 University Hospital of Patras, Patras, Greece4 “Agii Anargiri” Cancer Hospital, Athens, Greece5 “Henry Dunant” Hospital, Athens, Greece6 Hellenic Cooperative Oncology Group Data Office, Athens, Greece7 “Hygeia” Hospital, Athens, Greece8

ABSTRACT The combination of gemcitabine and gefitinib was evaluated in advanced pancreatic cancer. Totally, 53 patients were treated with a 7 week cycle of gemcitabine (1,000 mg/m2 given weekly) followed by six 4 week cycles of gemcitabine given on days 1, 8 and 15. Gefitinib 250 mg was administered daily. Responses were seen in 6, and stabilization of the disease in 12 patients. The main toxicity was myelotoxicity (92%). The 6-month progression-free survival (PFS) was 30%. Median PFS was 4.1 months and median survival 7.3 months with a 1 year survival rate of 27%. The above combination demonstrated promising activity in advanced pancreatic cancer.

INTRODUCTION Pancreatic adenocarcinoma is one of the most lethal cancers due to late stage of diagnosis. Patients with locally advanced, unresectable or metastatic disease have a grave prognosis, with 33,200 of 33,700 newly diagnosed cases dying of the disease in the USA alone (1). The 5 year survival rate is 4,000/μL, platelets >100,000/μL and hemoglobin >9.5 g/dL), hepatic function (alanine amino transferase and aspartate amino transferase 100 × 109 /L. For patients who did not achieve hematological recovery on day 22 of cycles 2–7, a complete blood count (CBC) was done twice a week, until the ANC was >2.0 × 109 /L and the platelet count >100 × 109 /L. In case the ANC was 0.5–2.0 × 109 /L and/or the platelet count was 50–100 × 109 /L, the dose of gemcitabine was reduced by 25%. In case the ANC was 75% tumor cells were considered to be eligible for genomic DNA extraction and

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sequence analysis, otherwise macro-dissection was performed to increase the tumor cell content. Tissue from 34 patients was available for the mutational analysis. All PCR’s, DNA sequencing and sequence confirmation were conducted as previously described (21). Exons 18, 19, 20, and 21 of the EGFR tyrosine kinase domain were amplified. K-RAS mutation analysis of codons 12 and 13 was performed using K-RAS specific primers amplifying exon 2 (22). An analysis of BRAF exon 15 was also performed, principally to determine the V600E status. Germline DNA was analyzed for all patients with suspected mutations in order to confirm mutations as somatic or germline in origin. Sequences were analyzed by BLAST chromatograms (manual review) and compared to reference gene sequence accession numbers. The EGFR exon 21 mutation L858R was also analyzed by PCR-RFLP based on the presence of a new Sau96I restriction site created by the mutation. Deletions in exon 19 were also analyzed by high performance gel electrophoresis (>2.5% agarose).

Statistical analysis The primary objective of the study was to evaluate the 6month PFS rate. According to Fleming’s single-stage design assuming that the expected 6-month PFS rate would be at least 30% and the minimum acceptable rate 15%, a total of 53 patients provided 80% power to test this hypothesis, with a 2-sided a = 5%. OS was measured from trial initiation to death or last followup, while PFS was measured from trial initiation to documented disease progression or death. OS and PFS distributions were evaluated using the Kaplan-Meier method and comparisons were performed by the log-rank test. Association between PTEN protein expression and tumor grade and presence of rash was evaluated using the Fisher’s exact test. In order to account for multiple comparisons Bonferroni adjusted p values were calculated and presented where appropriate.

RESULTS From June 2004 until May 2006, 54 patients entered the study. One patient did not receive any study medication and was not included in the analysis. Thirty-four of the 53 treated patients (64%) underwent some type of surgery before study entry (radical surgery 3, palliative surgery 4 and diagnostic surgery/biopsy 27 patients). None of the patients received radiation therapy prior to the study. Patient and tumor characteristics are shown in Table 1. In total, 9 patients completed 7 cycles of chemotherapy. Reasons for treatment discontinuation in the rest of the patients were disease progression (38 patients), voluntary withdrawal (4), toxicity (1), and death (1). The latter patient died at home, probably from cardiac arrest, after the sixth gemcitabine infusion of cycle 1. This patient did not have a history of cardiac disease and pretreatment ECG did not show any remarkable changes. Autopsy was not performed.

Table 1. Patient and tumor characteristics. Age Median Range Sex Men Women KPS 80–100% 60–70% ≤50% Grade I II III Unknown Extent of disease Locally advanced Metastatic CA 19-9 (U/mL) Median Range 50% of body surface), pruritus and fever. The symptoms persisted, even after appropriate treatment with antihistamines and antibiotics. This patient discontinued treatment with gefitinib after the 3rd week of the 1st cycle and continued treatment with gemcitabine monotherapy. In addition, in 9 patients (17%), gefitinib treatment was temporarily interrupted because of toxicity. Best response, as assessed by central review, was complete response in one patient (2%), partial response in 5 (9%) and stabilization of disease in 12 patients (23%). Main toxicities are depicted in Table 2. Myelotoxicity occurred in 50 patients (94%). Severe grade 3 anemia was observed in 2% of the patients, thrombocytopenia in 2%, leucopenia in 8%, and neutropenia in 15%, while one patient developed febrile neutropenia. G-CSF was administered in 13, erythropoietin in 26 and antibiotics in 8 patients. Furthermore, 4 patients had to be transfused with red blood cells, while no patient required platelet transfusion. Twenty-eight patients (53%) developed skin rash. After a median follow-up of 14.7 months (range, 0.1–19.6), all patients demonstrated disease progression and 46 (87%) had died. The 6-month PFS was 30%, while median PFS was 4.1 months [95% confidence interval (CI) 2.9–5.3] and median OS 7.3 months (95% CI 6.3–8.3) (Fig. 1). The one-year survival rate was 27%.

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Table 2. Incidence (%) of various toxicities. Anemia Leucopenia Thrombocytopenia Neutropenia Allergic reaction Pain (muscle) Pain (bone) Pain (other) Neurotoxicity (motor) Rash Nausea Vomiting Mucositis (oral cavity) Esophagitis Diarrhea Constipation Infection Alopecia Anorexia Fatigue Fever Salivary gland changes Deep vein thrombosis

Grade 1 45 34 21 13 2 8 6 15 2 43 23 8 8 0 21 15 0 8 25 26 13 0 0

Grade 2 34 19 4 28 2 2 0 8 0 9 13 8 6 2 15 6 11 8 9 13 15 2 0

Table 3. Biomarker profile. Grade 3 2 8 2 15 0 0 0 0 0 4 0 4 2 0 0 0 0 0 2 2 0 2 4

Grade 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Notably, in an unplanned retrospective analysis, patients with rash of any grade had significantly longer PFS (median 5.1 months, 95% CI 3.6–6.6) than those without rash (median 2.2 months, 95% CI 0–4.6, p = 0.002). However, the difference in OS between patients with and without rash was of borderline significance (median 8.8 months, 95% CI 4.6–13.0 vs. median

Figure 1. OS (median 7.3 months, red) and PFS (median 4.1 months, blue) for all patients included in the study.

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N(%) EGFR IHC n = 30 (NE 0) Positive Negative EGFR gene status n = 19 (NE 4) Normal Gain HER-2 IHC n = 28 (NE 2) Positive Negative HER-2 gene status n = 18 (NE 5) Normal Gain PTEN IHC n = 29 (NE 1) No Loss Loss PTEN gene status n = 20 (NE 3) Normal Deletion Gain K-RAS mutation n = 34 (NE 0) Wild Type Mutated EGFR mutation n = 34 (NE 0) Wild Type Mutated

23 (77) 7 (23) 16 (84) 3 (16) 6 (21) 22 (79) 14 (78) 4 (22) 7 (24) 22 (76) 5 (25) 14 (70) 1 (5) 17 (50) 17 (50) 32 (94) 2 (6)

NE = Non-Evaluable For each biomarker, n corresponds to the number of evaluable patients and percentages are calculated among them.

6.0 months, 95% CI 4.5–7.5, respectively, p = 0.059). The 1 year survival rates were 32% for those with rash, and 14% for those without rash, respectively, (p = 0.059). EGFR protein expression was found in 23/30 patients (77%), while EGFR gene gain was observed in 3/19 patients (16%) (Table 3). HER-2 protein expression (+2, +3) was observed in 6/28 patients (21%), while over-expression (+3) was seen in one patient. HER-2 gene gain was detected in 4/18 patients (22%) and PTEN gene deletion in 14/20 patients (70%) (Table 3). Representative IHC stains and FISH images for EGFR (a, b), HER-2 (c, d) and PTEN (e, f) are shown in Fig. 2. PTEN protein expression (no loss) was noticed in 7/29 patients (24%) and was the only biomarker associated with significantly longer PFS (Fig. 3). Median PFS was 10.6 months (95% CI 0.1–22.4 months) for patients with PTEN protein expression and 4.9 months (95% CI 2.1–7.6 months) for patients with PTEN protein loss (p = 0.009, Bonferroni adjusted p = 0.045). No association between the assessed biomarkers and OS was identified. PTEN protein expression was significantly associated with presence of rash in a retrospective analysis. All patients that had a PTEN protein-expressing tumor (7 out of 7), compared with half of the patients with PTEN protein loss (11 out of 22), developed rash during their treatment (p = 0.026). No association between PTEN protein expression and tumor grade was found. Mutational analysis of all gene exons examined was successful in all samples tested (Table 3). K-RAS mutations were identified in 17/34 patients (50%). No association between


Figure 2. a. EGFR protein expression, b. EGFR gene gain (red signals), c. HER-2 protein expression, d. HER-2 gene gain (red signals), e. PTEN protein loss, and f. PTEN gene deletion.


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Figure 3. OS (A) and PFS (B) for patients with PTEN protein expression (red) and PTEN protein loss (blue).

K-RAS mutations and OS, PFS, or any of the assessed biomarkers was identified. Two patients had an EGFR point mutation, both in exon 20 (Fig. 4). No BRAF exon 15 mutations were identified.

DISCUSSION The present study is one of the first to evaluate the activity of the combination of gemcitabine and gefitinib in patients with

Figure 4. Representative chromatograms (sense 5 and antisense 3 ) of EGFR exon 20 mutations (A. L815P, B. C797Y).

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advanced pancreatic cancer. On the other hand, experience with erlotinib, another TKI, in this disease is more extensive. In a pivotal phase III study conducted by the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG PA.3) (23), 569 patients with locally advanced or metastatic pancreatic cancer were treated with gemcitabine, in a similar to our study fashion, or gemcitabine combined with erlotinib, at a dose of 100 mg, as an initial step, or 150 mg given orally once a day. Median survival was significantly improved with the combination, from 5.91 months to 6.24 months, similar to that reported in our study. The 1 year survival rate was improved from 17% to 23% with the combination, which is very close to that found in our study. Following the results of this trial, erlotinib plus gemcitabine was approved in the USA and Europe for the treatment of advanced pancreatic cancer. The most common side effects with the addition of erlotinib were grade 1 and 2 rash (72%), diarrhea (56%) and stomatitis (23%). Experience with gefitinib in the treatment of pancreatic cancer is limited. Preclinical data have demonstrated increased activity of gefitinib in combination with docetaxel as compared to docetaxel alone. Initial results of a phase II study (24) with 31 patients enrolled showed that best response was stabilization of the disease seen in 5 patients. Median survival at the time of presentation of these results was 4.4 months. However, it has to be considered that previously treated patients were enrolled in this trial, as opposed to ours being a first-line treatment study, and that docetaxel is not thought to be as active in pancreatic cancer as other chemotherapeutic agents, such as capecitabine or oxaliplatin. Our results with the combination of gemcitabine and gefitinib appear to be promising. The 1 year survival of 27% is similar to that reported with gemcitabine and erlotinib (23) or other active chemotherapeutic combinations (6); however, response rates in the present study were low, similar to those seen in other trials with gemcitabine monotherapy or the combination of gemcitabine with other active agents (25, 26). Among our patients, toxicity from the addition of gefitinib was generally manageable. The most common SAEs were myelotoxicity and diarrhea. The rates of SAEs were similar to those recorded with gemcitabine and erlotinib (23). Rash from the administration of gefitinib was observed in 53% of our patients. Rash has been suggested as a surrogate marker of improved efficacy in patients with several tumor types treated with TKIs or EGFR-targeted monoclonal antibodies (27, 28), and this may also be the case in pancreatic cancer. In the NCIC study (23), even though analysis of OS based on EGFR status did not show a significant difference, grade of rash was associated with better outcome. Indeed, among patients with grade of rash ≥2, median OS was 10.5 months with a 1 year survival rate of 43%. In our study, patients with rash of any grade had significantly longer PFS than those without rash. There was no difference regarding OS. Even though a selection bias is possible, since, arguably, patients with rash would have lived longer and, thus, would have been exposed to the EGFR inhibitor for a longer period of time, one can not ignore the fact that rash in most cases peaks

in severity within the first 1 or 2 weeks of treatment and usually resolves by the second or third month. However, these results should be interpreted cautiously, since the number of patients is small for such unplanned analyses, and the existence of possible confounding factors cannot be excluded. An important part of our study was the evaluation of several biomarkers in relation to clinical outcome. A translational research part was also performed in the NCIC trial (23). EGFR in that study was tested centrally by IHC in 162 patients, with 53% of the samples being EGFR-positive (≥10% of tumor cells demonstrating membranous staining); however, EGFR protein expression was not found to affect survival. In our study, EGFR was assessed centrally by IHC in 30 patients. EGFR membranous staining, in >10% of tumor cells, was found in 77% of the samples, slightly higher than the 53% reported in the NCIC study (23). However, the range of EGFR expression by IHC in pancreatic ductal adenocarcinomas, reported in the literature, varies between 31% and 69% (29). The above results may be confounded by the fact that different antibodies for EGFR immunostaining and different interpretation methods were used in the studies. In line with the results of the NCIC trial, we did not find an association between EGFR protein expression and OS. HER-2 protein expression was observed in 6/28 (21%) cases, which is identical to the 21% reported in a pancreatic cancer tissue archive (30). We are currently evaluating several molecular markers using various techniques, such as PCR, FISH and IHC, in a large cohort of pancreatic tumor samples, in order to more accurately evaluate their incidence and prognostic significance in a Greek population with pancreatic cancer. An intriguing finding in the present study was that patients with tumors expressing the PTEN protein had longer PFS than those with PTEN protein loss. The PTEN gene is considered to be a key factor in human neoplasias (31). Somatic PTEN mutations have been identified in a variety of solid tumors, such as glioblastoma multiforme (32) and endometrial cancer (33). Most importantly though, aberrant PTEN expression has been implicated to contribute to the constitutive activation of the PI 3-kinase/Akt signaling pathway, resulting in loss of growth control of pancreatic cancer cells (34). These data are in complete agreement with our finding that pancreatic cancer patients with tumors expressing the PTEN protein have significantly longer PFS and support the notion that there might be value to the combined EGFR and PI 3-kinase/Akt suppression with IGF-1R inhibitors in the treatment of pancreatic cancer. PTEN loss, as a marker of resistance to gefitinib, has been suggested by several in vitro studies. She et al. (35) showed that resistance to gefitinib of EGFR over-expressing MDA-468 cells was attributed mainly to EGFR-independent constitutive Akt activation caused by loss of PTEN function in these cells. Additionally, restoration of sensitivity to gefitinib was observed following reconstitution of PTEN function through a tet-inducible expression system. Correlation of PTEN loss and resistance to EGFR TKIs has also been reported in several tumors, such as glioblastoma multiforme (36) and prostate cancer (37), while in


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non-small cell lung cancer (NSCLC), response to gefitinib or survival was not affected by PTEN protein expression (38). Considering that our patients were treated with gefitinib, we have analyzed molecular markers known to correlate with both response and resistance to TKIs, previously identified in NSCLC patients (20). In several studies, pancreatic patients have been identified with somatic mutations in the EGFR gene (39, 40). An exon 20 mutation was found in a patient with progressive disease (40), while an exon 19 delE746-A750 was found in 2 other patients with stable disease (39). This latter mutation has been correlated with response to TKIs in NSCLC patients (41). Both our patients harboring an exon 20 mutation, achieved at least the median PFS and OS. As K-RAS mutations are known to be mutually exclusive from EGFR mutations and a sign of resistance to TKIs (20, 40), K-RAS was also analyzed. Mutations were observed in 50% of our patients similar to the incidence reported by other studies (40). However, there were no significant differences in PFS or OS in patients with K-RAS mutations compared to those with wild type K-RAS. In conclusion, the present study indicates that the combination of gemcitabine and gefitinib has similar efficacy to that reported with gemcitabine and erlotinib. It should be noted, however, that the survival data from this study cannot be directly compared to the existing data and should be viewed as hypothesis generating rather than definitive evidence. Notwithstanding, the most important goal in the management of advanced pancreatic cancer remains the molecular identification of those patients who would benefit the most from chemotherapy or novel therapeutic interventions. As our study clearly indicates, patients with PTEN protein expression have a significantly longer PFS than those with PTEN protein loss, possibly due to increased sensitivity to gefitinib. This finding needs to be confirmed both in preclinical models and in prospective clinical trials in patients with pancreatic cancer.

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ACKNOWLEDGMENTS The authors wish to thank Ms. Georgia Vourli (MSc), HeCOG Data Office, Athens for the statistical analysis, Ms. Evita Fragou, Ms. Dimitra Katsala and Ms. Anna Tiga (AstraZeneca) for monitoring the study, Ms. Maria Moschoni, HeCOG Data Office, Athens for coordinating the data management, and Ms. Stella Dallidou for secretarial assistance. We are all grateful to our patients who had trusted us and participated in the trial.

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