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Background: We studied the combination of pemetrexed, a multi-targeted antifolate, and cetuximab, an mAb against the epidermal growth factor receptor, with radiotherapy in poor prognosis head and neck cancer.
Patients and methods: Patients received pemetrexed on days 1, 22, and 43 on a dose-escalation scheme with starting level (0) 350 mg/m2 (level −1, 200 mg/m2; level +1, 500 mg/m2) with concurrent radiotherapy (2 Gy/day) and cetuximab in two separate cohorts, not previously irradiated (A) and previously irradiated (B), who received 70 and 60–66 Gy, respectively. Genetic polymorphisms of thymidylate synthase and methylenetetrahydrofolate reductase were evaluated.
Results: Thirty-two patients were enrolled. The maximum tolerated dose of pemetrexed was 500 mg/m2 in cohort A and 350 mg/m2 in cohort B. Prophylactic antibiotics were required. In cohort A, two dose-limiting toxicities (DLTs) occurred (febrile neutropenia), one each at levels 0 and +1. In cohort B, two DLTs occurred at level +1 (febrile neutropenia; death from perforated duodenal ulcer and sepsis). Grade 3 mucositis was common. No association of gene polymorphisms with toxicity or efficacy was evident.
Conclusion: The addition of pemetrexed 500 mg/m2 to cetuximab and radiotherapy is recommended for further study in not previously irradiated patients.
Management of advanced head and neck cancer (HNC) represents a complex and challenging task that necessitates a multidisciplinary approach . Treatment with concomitant chemoradiotherapy has become the standard for selected patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN), conferring an absolute survival benefit of 6.5% at 5 years over radiotherapy alone [2, 3]. Although cisplatin, alone or with 5-fluorouracil, has been commonly used in chemoradiotherapy, the optimal regimen has not been established yet. The addition of novel targeted agents to radiotherapy has been explored in an attempt to further improve survival and reduce treatment-related toxicity.
Cetuximab, an IgG1 chimeric mAb against the extracellular ligand-binding domain of epidermal growth factor receptor (EGFR), was the first molecularly targeted agent to obtain regulatory approval in HNC as it provided improved locoregional control and survival when added to radiotherapy alone in a phase III randomized trial . There was no significant difference between the two arms regarding in-field toxic effects, such as mucositis and dermatitis; however, rash and hypomagnesemia were significantly more frequent with cetuximab. A main research focus has been to incorporate cetuximab or other novel agents into standard platinum-based chemoradiotherapy . However, cisplatin-based chemoradiotherapy is usually associated with multiple acute complications that make the addition of a second cytotoxic agent problematic due to the onset of dose-limiting toxicities (DLTs), including mucositis, neuropathy, nephrotoxicity and myelosuppression [1, 6]. Therefore, cetuximab-based combinations are attractive since this drug does not increase mucositis or hematologic toxic effects and may allow full doses of the chemotherapeutic to be delivered.
Pemetrexed is a novel multi-targeted antifolate that acts by inhibiting several key enzymes involved in nucleotide synthesis, such as thymidylate synthase (TS) . Functional gene polymorphisms of TS, the reduced folate carrier (SCL19A1), γ-glutamyl hydrolase and methylenetetrahydrofolate reductase (MTHFR) have been associated with outcome and toxic effects in patients receiving antifolates and may modulate pemetrexed activity and toxic effects as well [8–12]. Pemetrexed has been demonstrated to have clinical efficacy in phase III randomized trials in advanced non-small-cell lung cancer (NSCLC) [13, 14] and malignant mesothelioma . Phase II clinical trials of pemetrexed in recurrent or metastatic SCCHN reported promising activity [16, 17]. Moreover, pemetrexed has shown radiosensitizing properties across multiple tumor types in preclinical models [18–20]. There are limited clinical data with radiotherapy and pemetrexed, including a study in the re-irradiation setting after induction chemotherapy in patients with HNC . Also, a phase I trial evaluated chest radiotherapy with pemetrexed or pemetrexed and carboplatin in patients with thoracic malignancies . Our objective was to develop a novel non-cisplatin-containing chemoradiotherapy regimen by incorporating pemetrexed into standard radiotherapy and cetuximab (‘XPemE’ regimen) for the treatment of patients with advanced HNC.
Patients ≥18 years with advanced HNC who required radiotherapy to the head and neck were eligible. Other key eligibility criteria included performance status of zero to two, according to the Eastern Cooperative Oncology Group scale, absolute neutrophil count (ANC) ≥1500/mm3, hemoglobin >8 g/dl, platelet count ≥100000/mm3, total bilirubin levels within normal limits, liver transaminases not exceeding three times the upper limit of normal, and creatinine clearance ≥45 ml/min. Any number of prior systemic therapies was allowed, except prior treatment with EGFR inhibitors or pemetrexed. Patients with metastatic disease were eligible if they had predominant symptoms from locoregional disease and required radiotherapy; patients with newly diagnosed SCCHN were eligible if they had stage IV disease and poor expected survival. The study protocol was approved by the University of Pittsburgh Institutional Review Board and all patients provided written informed consent before study entry. The trial was registered with clinicaltrials.gov (NCT00291707).
One week before starting radiotherapy, cetuximab (supplied by Bristol-Myers Squibb, Princeton, NJ) 400 mg/m2 was i.v. administered over 2 h. Subsequent cetuximab infusions were given at a dose of 250 mg/m2 over 60 min once weekly during the 6–7 weeks of radiotherapy. Pemetrexed (supplied by Eli Lilly and Company, Indianapolis, IN) was administered i.v. over 10 min every 21 days, on days 1, 22 and 43 (following cetuximab when both drugs were given on the same day). The pemetrexed dose on day 43 was only given if the total radiotherapy dose exceeded 60 Gy. Cetuximab was not withheld for known pemetrexed toxic effects. All patients received folic acid 1000 μg once daily orally and vitamin B12 1000 μg i.m. every 9 weeks beginning 1 week before starting pemetrexed and dexamethasone 4 mg twice daily orally, 1 day prior, the day of, and 1 day after pemetrexed. Premedication with 50 mg diphenhydramine hydrochloride or an equivalent antihistamine was administered 30–60 min before cetuximab.
Radiation was delivered with intensity-modulated radiation therapy in all cases, planned with the Eclipse planning system (Varian Medical Systems, Palo Alto, CA). Tumor and planned treatment volumes were identified via a computed tomography (CT) simulation (in most cases, combined positron emission tomography (PET)–CT was used). Patients were treated in the supine position, with Aquaplast masks (WFR/Aquaplast Corp., Avondale, PA) for immobilization. Radiotherapy was administered once daily, 2 Gy per fraction, 5 days a week. In the not previously irradiated cohort, the entire neck was irradiated with a total dose of at least 46–50 Gy. High-risk nodal regions received 60 Gy. The total dose to the primary tumor and clinically positive nodes was 70–74 Gy. The maximum dose permitted to the spinal cord did not exceed 45 Gy. For previously irradiated patients (cohort B), smaller fields were used that were limited to the involved area, i.e. adjacent high-risk area plus a 1.5–2 cm margin, and the total radiation dose was up to 66 Gy (minimum 60 Gy). In this cohort, the cumulative spinal cord dose was not to exceed 50 Gy.
All supportive measures consistent with optimal patient care were provided throughout the study. Prophylactic administration of granulocyte colony-stimulating factor was not permitted. The use of recombinant erythropoietin was discouraged and transfusions were recommended if hemoglobin dropped <10 g/dl during radiotherapy. Prophylactic placement of a gastrostomy tube (G-tube) was at the discretion of the treating physician but it was strongly recommended for patients with significant baseline dysphagia and weight loss. After the first 11 patients were enrolled in group A, two episodes of neutropenic fever with ANC <500/mm3 and an episode of fever with ANC >500/mm3 were recorded. Thus, subsequent patients received prophylactic ciprofloxacin 500 mg twice daily or levofloxacin 500 mg once daily orally during the second and third cycle of pemetrexed for 10 days (days 4–13 of each cycle).
Before initiating treatment, patients were evaluated with history and physical examination, including dental and otolaryngology examination, electrocardiogram, and blood samples for correlative studies. Swallowing assessments were carried out as standard practice at baseline and then as indicated. History and physical examination, performance status, complete blood counts (CBC), and serum chemistry profile, including electrolytes and liver function tests, were assessed at baseline and before each pemetrexed cycle (i.e. every 3 weeks). During treatment, clinical evaluation, CBC, serum electrolytes, including magnesium, were carried out weekly. Toxic effects were recorded and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. Radiological evaluation by CT, magnetic resonance imaging and/or PET scans was done for baseline tumor assessment and in ~8 weeks after completion of radiotherapy. RECIST  was used for tumor response assessment. Subsequently, repeat imaging with CT scans of chest and head and neck was carried out every 6 months for 3 years. Biopsies of recurrent lesions were carried out as clinically indicated. Patients were followed every 3 months for 2 years and then every 6 months for 3 years and subsequently annually for up to 10 years.
The primary objective of this phase I trial was to evaluate the DLTs and determine the maximum tolerated dose (MTD) of pemetrexed in combination with radiotherapy and cetuximab. DLT was defined as any of the following during treatment and 3 weeks of follow-up: grade 4 neutropenia >5 days or febrile neutropenia with an ANC <500; grade 4 stomatitis, mucositis, dermatitis and/or dysphagia that lasted >five consecutive days; grade 4 thrombocytopenia; grade 4 nausea or vomiting despite appropriate antiemetic therapy; grade 4 rash; any other grade 3 or higher non-hematologic toxicity, except for anorexia, fatigue, infection without neutropenia, grade 3 elevation of transaminases, and grade 3 hypomagnesemia; and delays in treatment due to toxicity of >3 weeks. Patients who developed grade 3 or 4 infusion reaction were removed from the study and replaced without this counted as a DLT.
Accrual and dose escalation was conducted separately in groups A and B. Dose levels were escalated in cohorts of three to six patients, starting with dose level 0, on three dose levels. We employed a variant of the ‘3+3’ design that allowed for up to six patients to be treated on each dose level. While dose escalation may occur based on none of the three patients experiencing DLT at dose level 0, at the same time one to three additional patients may be receiving treatment at dose level −1. This variant was used to speed up the process due to lengthy evaluation of toxicity (duration of radiotherapy plus 3 weeks of follow-up, i.e. up to 9–10 weeks). Once the MTD was determined, three to six additional patients were to be enrolled at that dose level in order to further characterize treatment-related toxic effects. The secondary objectives of this study include the objective response rate, locoregional control, overall survival (OS) and progression-free survival (PFS). Survival data were analyzed using the Kaplan–Meier method and compared using the log-rank test. We also carried out an exploratory analysis for an association of MTHFR and TS polymorphisms with grade 3–4 toxic effects and PFS.
Blood samples for genotyping were collected at baseline. DNA was isolated using Gentra Systems Inc. (Minneapolis, MN) DNA isolation kits. DNA quantity and quality was assessed using the Thermo Scientific NanoDrop 1000 full-spectrum UV/Vis spectrophotometer. The MTHFR (rs1801133, rs1801131 and rs2274976) was detected using custom TaqMan-based single nucleotide polymorphism (SNP) genotyping kits from Applied Biosystems, run on the ABI Prism 7700 Sequence Detection systems v 1.7 (Applied Biosystems, Foster City, CA) . The TS gene promoter repeat and SNP (2 or 3 repeats; G>C within second repeat of the 3R allele) polymorphisms were detected by PCR and restriction fragment length polymorphism–PCR analyses as previously described . Positive and negative PCR controls were included with each amplification reaction. Samples were genotyped in a blinded fashion and an additional 10% of samples were repeated to verify the reproducibility of the assay. All results were interpreted independently by two laboratory personnel.
From March 2006 to February 2008, 32 patients with advanced HNC were enrolled in the study: 23 in cohort A and 9 in cohort B (Table 1). Twenty-five patients (78%) had squamous cell carcinoma. Twenty patients had newly diagnosed tumors, nine patients had recurrent tumors and three patients had a second primary cancer. Thirty-one patients were assessable for toxicity and 30 for DLT, while 2 patients were replaced (Table 2); one patient in cohort A withdrew consent due to transportation issues on the second week of treatment and was not considered assessable for toxicity; another patient in cohort A had a sudden death probably from aspiration that was not considered treatment related.
Among 23 patients in cohort A, 19 received three cycles of pemetrexed (83%), 3 patients received two cycles and 1 patient (who withdrew from study) received one cycle. In cohort B, eight patients (89%) received three cycles and one patient two cycles. A total of five patients (16%) were not able to complete the planned cycles of pemetrexed. In cohort A, 1 patient received nine weekly doses of cetuximab, 14 patients received eight doses, 4 patients seven doses, 3 patients four to six doses, and 1 patient one dose. In cohort B, eight patients received eight weekly doses of cetuximab and one patient seven weekly doses. The radiation doses delivered (median [range]) were 70 Gy (38–74 Gy) in cohort A and 66 Gy (60–70 Gy) in cohort B. All but three patients in cohort A received at least 70 Gy (six of these patients had delays in radiotherapy of 2–10 days); one patient discontinued radiotherapy at 40 Gy due to dose-limiting febrile neutropenia and grade 4 mucositis, one patient had sudden death after receiving 38 Gy, and one withdrew from the study on the second week of treatment without developing significant toxic effects. In cohort B, all but one patient, who developed neutropenic fever, received 66 Gy.
Five patients experienced a DLT (Table 2). In cohort A, one patient on each dose level of pemetrexed developed febrile neutropenia with ANC <500/μl. An additional patient at dose level −1 developed fever with ANC >500/μl, which did not meet criteria for DLT. Therefore, dose level +1 (500 mg/m2) was determined as the MTD of pemetrexed for not previously irradiated patients. In cohort B, two patients, both at pemetrexed dose level +1, experienced a DLT. One had febrile neutropenia, whereas the other presented with perforated duodenal bleeding and subsequent grade 5 sepsis with grade 4 thrombocytopenia and neutropenia. Hence, the MTD of pemetrexed for previously irradiated patients was determined at 350 mg/m2.
Hematological toxic effects are shown in Table 3. In cohort A, six patients developed grade 3 and five patients grade 4 neutropenia. In cohort B, three patients experienced grade 4 neutropenia. Serious thrombocytopenia was rare; one patient in group A and two patients in group B developed grade 3–4 thrombocytopenia.
Mucositis, dysphagia, rash and dermatitis were the most common non-hematologic toxic effects (Table 4). Sixteen patients developed grade 3 mucositis and one patient (at dose level 3) developed grade 4 mucositis. In cohort A, the rate of grade 3–4 mucositis was 4 of 7 (57%), 5 of 7 (71%) and 8 of 9 (89%) at dose levels −1, 0, and +1, respectively. Gastrostomy tube was placed in six patients during treatment, whereas seven patients had G-tube placed before treatment initiation. Five patients remained G-tube dependent upon last follow-up; however, only one of them in cohort B, who had G-tube before treatment, was also progression free. Nine patients developed infection with the most common being aspiration pneumonia (four patients). Thirteen patients developed grade 1 and two grade 2 hypomagnesemia that was reversible. All patients developed cetuximab-associated skin rash of any grade, and among them only four patients had grade 3 rash.
Of 21 patients assessable for response in cohort A, 12 patients (4 on each dose level) achieved a complete response and 7 a partial response (4 at dose level +1; 2 at dose level 0; 1 at dose level −1). Of seven assessable patients in cohort B, four patients achieved a complete response (three at dose level 0; one at dose level +1) and three a partial response (two at dose level 0; one at dose level +1). With a median follow-up of 36 months, the 3-year PFS was 41% in cohort A and 22% in cohort B, and the 3-year OS was 55% and 37%, in cohorts A and B, respectively. Two patients died during treatment; one death was possibly treatment related. One additional patient developed severe neck fibrosis and facial edema and died without documented disease progression 8 months after treatment completion. Among 13 patients with locally advanced stage IV SCCHN (5 with an oropharyngeal primary, 4 laryngeal, 2 hypopharyngeal, and 2 with unknown primary) who completed radiotherapy (an additional patient who did not complete radiotherapy progressed and died), 6 patients (46%) remained progression free but only 2 recurred locoregionally (i.e. 85% locoregional control) with a median follow-up of 33 months. Seven of these 13 patients had available tumor specimens that were tested for human papillomavirus (HPV) by in situ hybridization and p16 by immunohistochemistry; all but 1 patient had HPV- and p16-negative tumors (the patient with HPV-/p16-positive tumor had a base of tongue primary and developed disease progression in mediastinal lymph nodes). A patient with anaplastic thyroid cancer and a patient with medullary thyroid carcinoma remained progression free after 32 and 36 months of follow-up, respectively.
Twenty-eight patients underwent genotyping for MTHFR and TS. The frequency of the MTHFR and TS polymorphisms are shown in Table 5. The genotype frequencies were in Hardy–Weinberg equilibrium. Polymorphisms in MTHFR and TS did not correlate with severity of treatment-related toxic effects (data not shown). Furthermore, the different variants of MTHFR and TS did not seem to affect either the response rate or the survival.
We evaluated a novel regimen with the addition of pemetrexed to radiotherapy and cetuximab for HNC. This is one of the first reports of pemetrexed as a radiosensitizer for the treatment of HNC. Villaflor et al.  reported preliminary data in the re-irradiation setting using a regimen with radiotherapy and concurrent carboplatin and pemetrexed after induction chemotherapy with gemcitabine and pemetrexed. In our study, dose escalation was carried out separately in previously irradiated and non-irradiated patients and phase II recommended doses of pemetrexed were established for each cohort. Grade 3–4 neutropenia was common (50% in cohort A and 33% in cohort B) and febrile neutropenia was the most frequent DLT that necessitated the addition of prophylactic antibiotics. Grade 3–4 mucositis was frequently observed (seven cases of grade 3 and one case of grade 4 in nine patients treated at the MTD in cohort A); however, a minority of patients required a G-tube and delays of radiotherapy were infrequent. Of 13 patients with stage IV SCCHN (5 of whom had an oropharyngeal primary) treated with a curative intent and completed radiotherapy per protocol, only 2 relapsed locoregionally (85% locoregional control), which suggests that pemetrexed is a potentially efficacious radiosensitizer.
The toxicity profile of XPemE differs from that of cisplatin-based chemoradiotherapy for HNC. XPemE was not associated with common acute and late toxic effects seen with cisplatin (e.g. neuropathy, ototoxicity, nephrotoxicity); however, neutropenia and mucositis were common. The incidence of mucositis with this regimen is comparable with other aggressive multi-agent regimens, such as radiotherapy, 5-fluorouracil, hydroxyurea-based regimens [26, 27]. Expected toxic effects with single-agent pemetrexed include myelosuppression with rare incidence of febrile neutropenia and elevation of transaminases; however, the development of neutropenia as predominant toxicity in our study was somewhat surprising. With the use of folic acid and B12, pemetrexed toxic effects are ameliorated . The combination of pemetrexed and cetuximab has been studied in a phase I/II trial in advanced NSCLC . Dose escalation of pemetrexed to 750 mg/m2 was feasible and in combination with standard weekly cetuximab dosing was well tolerated and not associated with an excessive incidence of neutropenia or febrile neutropenia. Also, the combination of radiotherapy and cetuximab is not known to result in febrile neutropenia . A Cancer and Leukemia Group B study investigated the combination of chest radiotherapy, carboplatin and pemetrexed with or without cetuximab . Twenty percent and 35% of patients had squamous histology in the arm with and without cetuximab, respectively, which may have affected efficacy results since pemetrexed has inferior activity in squamous cell NSCLC. Febrile neutropenia was experienced by 5% and 7% of patients in arms A and B, respectively. The high incidence of neutropenia, and especially febrile neutropenia, in our study may be attributed to the potent radiosensitizing properties of pemetrexed that resulted in bone marrow suppression as well as the mucosal barrier injury caused by mucositis. Villaflor et al. reported myelosuppression, infectious complications and other considerable toxic effects with re-irradiation with concurrent carboplatin and pemetrexed after induction chemotherapy with gemcitabine and pemetrexed, which mirrors observations from the same and other groups of investigators with various chemoradiotherapy regimens in the re-irradiation setting [21,30–32]. Despite using smaller fields of radiotherapy in previously irradiated versus radiation-naive patients, we could not escalate pemetrexed dose beyond 350 mg/m2 in this group of patients. The clinical relevance of gene polymorphisms in folate metabolism enzymes is uncertain. We looked at pemetrexed drug pathway-associated gene polymorphisms, specifically MTHFR and TS, and their possible association with toxicity and efficacy in patients with HNC receiving pemetrexed with cetuximab plus radiotherapy. This exploratory analysis did not reveal any significant associations, possibly because of the small sample size.
Pemetrexed is being studied in recurrent or metastatic SCCHN, including a phase II trial by our group that showed encouraging survival data . Further evaluation of XPemE in the phase II setting is recommended. We are currently investigating XPemE in a phase II randomized trial in patients with locally advanced SCCHN (NCT00703976).
Lilly Oncology; Bristol-Myers Squibb; National Cancer Institute Head and Neck Cancer Specialized Program of Research Excellence Grant (P50 CA097190-06).
AA has received research support from Lilly Oncology and AA, RLF, and JRG from Bristol-Myers Squibb. All remaining authors have declared no conflicts of interest.
Preliminary results of this study were reported at the 2008 meeting of the American Society of Clinical Oncology.