Early tumor progression associated with enhanced EGFR signaling with bortezomib, cetuximab, and radiotherapy for head and neck cancer

Abstract

PURPOSE

A phase I clinical trial and molecular correlative studies were performed to evaluate preclinical evidence for combinatorial activity of proteasome inhibitor bortezomib, epidermal growth factor receptor (EGFR) inhibitor cetuximab, and radiation therapy.

EXPERIMENTAL DESIGN

Patients with radiotherapy-naïve stage IV or recurrent squamous cell carcinoma of the head and neck (SCCHN) were studied. Escalating doses of bortezomib (0.7, 1.0 and 1.3 mg/m²) were given intravenously twice weekly on days 1, 4, 8, 11, every 21 days, with weekly cetuximab beginning 1 week prior and concurrently with intensity modulated radiotherapy (IMRT), delivered in 2Gy fractions to 70-74 Gy. Molecular effects were examined in serial serum and SCCHN tumor specimens, and the cell line UMSCC-1.

RESULTS

Seven patients were accrued before the study was terminated when 5/6 previously untreated patients with favorable prognosis oropharyngeal SCCHN progressed within 1 year (PFS =4.8 months; 95% CI, 2.6-6.9). Three patients each received bortezomib 0.7 or 1.0 mg/m², without dose-limiting toxicities; 1 patient treated at 1.3 mg/m² was taken off study due to recurring cetuximab infusion reaction and progressive disease. Expected grade 3 toxicities included radiation mucositis (n=4), dermatitis (n=1), and rash (n=1). SCCHN-related cytokines increased in serial serum specimens of patients developing progressive disease (P=0.029). Bortezomib antagonized cetuximab- and radiation-induced cytotoxicity, degradation of EGFR, and enhanced prosurvival signal pathway activation in SCCHN tumor biopsies and UMSCC-1.

CONCLUSIONS

Combining bortezomib with cetuximab and radiation therapy demonstrated unexpected early progression, evidence for EGFR stabilization, increased prosurvival signaling and SCCHN cytokine expression, warranting avoidance of this combination.

Introduction

Epidermal growth factor receptor (EGFR) is upregulated in many cancers, including approximately 90% of squamous cell carcinomas of the head and neck (SCCHN), where it is associated with decreased patient survival.^1,2 Cetuximab (ERBITUX™) is a humanized chimera of C225 that is FDA-approved for use in combination with radiation for treatment of SCCHN. A phase III clinical trial showed that the addition of cetuximab to radiotherapy (RT) results in an approximately 10% improvement in survival over RT alone in patients with locally advanced SCCHN, particularly those of the oropharynx.³ EGFR is implicated in cellular transformation, cell-cycle progression, DNA repair, prosurvival signal pathway activation, and angiogenesis. ^4-8 Inhibition of EGFR by anti-EGFR monoclonal antibody C225 has been shown to block pathways leading to inhibition of tumorigenesis and sensitization of EGFR driven tumors. Resistance of remaining SCCHN to EGFR inhibitors has been attributed to EGFR overexpression, mutations, or EGFR-independent mechanisms that co-activate multiple signal pathways important for cancer cell survival.^{1,2, 9-13}

Several prosurvival pathways have been reported to be variably activated by EGFR and other signals in SCCHN, including the Mitogen-Activated Protein Kinases (MAPKs), AKT, Nuclear Factor-kappa B (NF-κB), and Signal Transducer and Transcription (STAT)-3 pathways.^8-11 Among these, studies using SCCHN cell lines have revealed that aberrant signaling by cytokine and other growth factor pathways mediate EGFR-independent activation of NF-κB.^9,12 NF-κB is a key family of signal-activated transcription factors that affect prosurvival gene activation, the malignant phenotype, and prognosis.¹² Bortezomib (VELCADE™, PS-341) is an inhibitor of the 26S proteasome, a macromolecular complex important in degradation of proteins, including Inhibitor-κBs, that can block activation of NF-κBs.¹⁴ In preclinical and phase I studies, bortezomib was shown to inhibit NF-κB activation and have cytotoxic, anti-angiogenic, and radiosensitizing activity in SCCHN and other tumors. ^15-18 However, in combination with re-irradiation, bortezomib showed limited clinical activity, and lacked the ability to inhibit activated components of the EGFR-inducible MAPK and STAT3 pathways.^18-19 Together, preclinical and clinical results suggested that EGFR inhibitor-dependent signal pathways, and NF-κB proteasome-dependent pathways, are independently activated and contribute to the malignant phenotype and clinical response of SCCHN. ^{8,9, 19} Combined treatment with either of these agents individually with radiation, or with proteasome and EGFR inhibitors, had cytotoxic activity in preclinical and/or early phase clinical studies.^{1, 2, 16, 17, 20-24}

We conducted a phase I study to examine the effects of combination of bortezomib-proteasome and cetuximab-EGFR inhibition with intensity modulated radiation therapy (IMRT) in patients with advanced SCCHN. The primary objectives included evaluation for the toxicities and the maximum tolerated dose (MTD) of this combination. Secondary objectives included clinical response, progression-free and overall survival. Correlative studies evaluated the effects of combined bortezomib and cetuximab to inhibit activation of the EGFR, MAPK, AKT, STAT3 and NF-κB signal pathways, tumor cell survival, and levels of pro-inflammatory and angiogenic cytokines regulated by these pathways and detectable in serum of patients with SCCHN.

Methods

Patient selection

Protocol NCI-7893 was conducted at the National Institute of Health and the University of Pittsburgh, after obtaining approval by the respective Institutional Review Boards and informed consent. Eligibility criteria included age≥18 years; pathologically confirmed SCCHN or poorly/undifferentiated carcinoma of any head/neck site except the nasopharynx; previously untreated stage IV disease, residual disease or regional recurrence, without or with distant metastatic disease <3cm; ECOG performance status 0-1; adequate organ function; recovery from any prior surgery or chemotherapy including prior cisplatin >3 months; and no prior systemic EGFR inhibitors, bortezomib, head and neck radiation, uncontrolled intercurrent illness; or grade ≥2 peripheral sensory neuropathy.

Treatment Plan and Patient Assessments

The schema for the treatment plan and correlative studies is shown in Fig. 1. A standard 3+3 dose escalation design (3 subjects without, or up to 6 subjects after a dose limiting toxicity per dose level) was planned. Bortezomib (0.7, 1.0 and 1.3 mg/m²) was given intravenously twice weekly on days 1, 4, 8, 11, every 21 days. To obtain serum and optional tumor biopsies with the drug combination without and with radiation, bortezomib and cetuximab 400 mg/m² were started 1 week prior to combining bortezomib and weekly cetuximab 250 mg/m² with intensity modulated radiation therapy. Tumor received 2Gy per fraction once daily 5 days per week to 70-74Gy. Regions of intermediate and low risk were received 60-64 and 50 Gy, respectively. Bortezomib (Millennium Pharmaceuticals Inc, Cambridge, MA) was provided through a Clinical Trials Agreement, Cancer Therapeutics Evaluation Program, National Cancer Institute (NCI).

Figure 1. Dosing Schema NCI 7893 Bortezomib with weekly Cetuximab and IMRT.

Patients were given escalating doses of Bortezomib (0.7, 1.0 and 1.3 mg/m²), twice weekly by IV on days 1, 4, 8 and 11 every 3 weeks. Bortezomib and cetuximab (400mg/m² loading dose, bold arrow) were started during week 1, followed by bortezomib and weekly cetuximab (250 mg/m², non-bold arrows) concurrent with IMRT 2Gy/day 5 days per week to 70-74 Gy. Serum was collected as indicated for SCCHN-related cytokines and optional tumor biopsies were obtained prior and during the first cycle of treatment as indicated.

Baseline evaluation included history, physical exam, standard laboratory tests, and CT or CT-PET imaging of the head, neck and chest obtained within 2 weeks of treatment. During treatment, patients underwent weekly physical exam, toxicity evaluation, CBC and blood chemistries. Toxicities were assessed by NCI common terminology criteria for adverse events (http://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/ctcaev3.pdf). Collection of serum was planned pretreatment and during each cycle, and optional tumor biopsies were planned pretreatment and week 1 and 2 of cycle 1 as in Fig. 1. Tumor measurements were performed at baseline, 2 and 5 months after completion of radiotherapy. Response and progression was evaluated using RECIST criteria.²⁵

Study Endpoints

The primary endpoints included dose limiting toxicities (DLTs), other toxicities, and the maximum tolerated dose (MTD) of bortezomib for this combination regimen. Patients were evaluable for toxicity if they received one cycle of therapy, or if they had DLT during the first cycle. Evaluation for DLT included the period on drug/radiation treatment plus 4 weeks of follow-up. DLTs were defined as CTCAE 3.0 grade 4 toxicities for: in-field stomatitis/mucositis, dermatitis or dysphagia lasting >5 days; rash; nausea/vomiting despite appropriate antiemetic therapy; absolute neutropenia <500/μL for more than 7 days, or neutropenic fever; thrombocytopenia; and recurrent grade 4 hematologic toxicities following delay or dose modification. Other DLTs included grade 3 or recurrent grade 2 neuropathy despite dose delay or modifications; all other grade 3 or higher toxicities, except grade 3 fatigue; infection without grade 4 neutropenia; in-field toxicities and nausea/vomiting as above; weight loss; dehydration; creatinine; hypotension; anorexia; pain; and any grade hypomagnesemia, hypokalemia or hyponatremia. DLT also included treatment delay due to toxicity of more than 3 weeks, except cetuximab infusion reactions ≥ Gr3, for which study removal and replacement were planned. Toxicities attributable (possible, probable or definite) to the study treatment were used for determination of DLT and MTD.

Secondary clinical endpoints included objective response, progression-free survival and overall survival. Secondary correlative endpoints included pre- to post-treatment changes in a set of serum and tumor biomarkers below.

Serum Cytokine and Growth Factor Assays

Concentrations of serum cytokine and growth factors were determined as described previously.^{18, 26} Peripheral blood sample collection was planned within 2 weeks of initiation of treatment, and then after initiation of study drugs on days 1, 5 and 12 of the first cycle. Thereafter, optional blood for serum could be collected on day 1, 5 and 12 of the second and third cycles (weeks 5 and 8) of bortezomib; following completion of RT at 8 weeks; and up to 3, 6, 12, 15, 18, 21 and 24 months. See Supplemental Methods for details.

Correlative studies of SCCHN tumor and cell line UMSCC-1

Serial SCCHN tumor biopsies were obtained pretreatment, on day 5 after induction with bortezomib and cetuximab, and on day 12 after combination with IMRT from one patient (#7) who consented to optional biopsies. To confirm and elucidate the mechanism of results obtained, HNSCC cell line UM-SCC1 was treated with bortezomib, cetuximab and/or radiation. Methods for immunoblotting of SCCHN tumor and UMSCC-1^13,17 and clonogenic survival assays¹⁷ for correlative studies were described previously, and as modified in Supplementary Methods.

Statistics

Using the standard 3+3 design, dose escalation is based on a ≤33% true rate of DLT in 3 patients, and MTD on a ≤16% true rate if the cohort is expanded to 6 subjects for a DLT.¹⁸ Based on previous studies linking increasing cytokine levels with progressive disease,²⁶ an exploratory comparison of the progression free survival between patients whose early cytokine changes after initiating treatment tended to increase vs. those whose values tended to decrease or remain steady was done using an exact log-rank test. For clonogenic survival assays, the difference in the surviving fraction after combination of drug treatments and 2 Gy irradiation was compared, as the surviving fraction in cell lines has been reported to correlate with the radiocurability of the corresponding human tumors in vivo.²⁷

Results

Patient characteristics, treatment and response

Subjects (Table 1) included 6 previously untreated patients with stage IV and 1 patient who presented after incomplete staging and surgical neck dissection with recurrent tonsil and neck SCCHN; 6 had oropharyngeal and 1 had a laryngeal primary. Treatment delivery (Supplemental Table 1) was completed in the first 6 patients, although bortezomib dose reduction was required in one dose level 2 patient for thrombocytopenia. Patient 7 (1.3 mg/m² dose level) was taken off study after 8 doses of bortezomib and 6 doses of cetuximab for recurrent grade 2 cetuximab infusion reactions and progressive disease (PD). There were no DLTs and a MTD for combination of bortezomib with cetuximab and radiation was not reached before the study was ended. Clinical outcomes (Table 2) precipitated termination of the study after 5 out of 6 of the previously untreated patients exhibited progression within one year. Only 3 of 7 patients achieved a complete response (CR) within 2 to 5 months (patients 1,4,6). Of these, patient 4 developed a solitary pulmonary metastasis at 11 months. Three patients had PD during (patient 7) or within 5 months of treatment (patients 3, 5). Overall, 3 had local-regional and 2 had pulmonary PD. Local-regional failures occurred within the 70Gy treatment region.

Table 1. Patient and tumor characteristics.

Patient number	Age (years)	Gender	Stage/ resectability	Primary site	HPV & p16 status	Smoking history	Alcohol history
1	52	Male	Original TxN2aM0, Recurrence tonsil, neck resectable	Tonsil	N/A	Former 35pk/yrs	No
2	48	Male	T3N2cM0 resectable	Base of tongue	HPV+	Current 68pk/yrs	No
3	58	Male	T1N2aM0 resectable	Base of Tongue	HPV+	Never	Yes
4	62	Male	T4N2cM0 resectable	Base of Tongue	HPV − / p 16+	Former weekly pipe/cigar	Yes
5	61	Male	T3N2cM0 unresectable	Base of Tongue	HPV+	Former 48pk/yrs	Yes
6	50	Female	T3N2cM0 resectable	Larynx	HPV−	Former 35pk/yrs	Yes
7	54	Female	T1N2aM0 resectable	Tonsil	HPV+/p16+	Current 30pk/yrs	Yes

Abbreviations: TNM, Tumor, Node, Metastasis; Pk/yrs=cigarette pack years; N/A, not available Patient 1 presented with incomplete TxN2aM0 staging and treatment consisting of neck dissection only, after which he sought care with recurrent tonsil and neck disease

Table 2. Treatment and outcomes.

Patient number	Bortezomib dose level (per m2)	Best objective response	Disease progression	Progressionfree interval (months)	Site of progression	Salvage therapy	Overall survival (months)	Survival status
1	0.7 mg/m2	CR	No	14	None	None	14	NED
2	0.7 mg/m2	PD	Yes	6	Distant	None	8	DOD
3	0.7 mg/m2	PD	Yes	5	Regional	Neck surgery	17	NED
4	1 mg/m2	CR	Yes	11	Distant	Lobectomy	23	NED
5	1 mg/m2	PD	Yes	5	Local	None	17	DOD
6	1 mg/m2	CR	No	24	None	None	24	NED
7	1.3 mg/m2	PD	Yes	1	Regional	Chemoradiotherapy	18	NED

Best response by CT/PET at 2 or 5 months post-treatment: CR, complete response; PD, progressive Disease; Clinical Progression-Free and Overall Survival as of last visit or 12/1/10; NED, no evidence of disease; DOD, dead of disease

The median Progression Free Survival (PFS) was only 4.8 months (95% CI, 2.6-6.9), including early recurrence in 5/6 with previously untreated oropharynx cancer, which compared unfavorably with a median PFS of 17 months reported for cetuximab and radiation at that site.³ While stratification for tumor site and testing for human papillomavirus (HPV) or p16 status were not incorporated in design of this phase I study, of six patients with oropharyngeal primary site lesions, patients 2, 3 and 5 were reportedly HPV+ prior to study entry, and specimens from PD lesions in patients 4 (lung) and 5 (oropharynx) tested p16+ at the site of progressive disease, consistent with HPV origin. Of the 5 with PD, patients 2, 4, 5, and 7 had other co-factors considered to increase risk of recurrence (T stage≥3 or unresectable; N stage≥2; and current or former smoking ≥10 pack/years). Early detection of PD and salvage therapy by parotidectomy (patient 3), lobectomy (patient 5), or cisplatin concurrent with remaining radiation (patient 7) achieved disease-free status in 3/5 recurrent patients, who together have a median overall survival of 18 months at last follow-up. The patient with a laryngeal primary and CR remains disease-free after 24 months.

Toxicities

Toxicities (Table 3) included expected grade 3 toxicities for the treatment combination, such as mucositis (4), dysphagia (3), xerostomia (1), and dermatitis (1); cetuximab-associated acneiform rash (1); and bortezomib-associated peripheral neuropathy (1). One Grade 3 infection occurred in a patient without neutropenia.

Table 3. Worst toxicities (n=7).

	Grade 0	Grade 1	Grade 2	Grade 3	Grade 4
Neutropenia	6	1	0	0	0
Anemia	3	3	1	0	0
Thrombocytopenia	5	0	2	0	0
Lymphopenia	4	0	0	1	2
Infection with normal or grade1/2 neutrophils	5	0	0	2	0
Infection, other	5	0	2	0	0
Mucositis/stomatisis (clinical exam)	0	0	3	4	0
Dysphagia	0	0	3	4	0
Radiation dermatitis	0	1	2	4	0
Rash – Acneiform	4	0	2	1	0
Rash – Desquamation	5	1	1	0	0
Dermatology-Other	5	1	1	0	0
Allergic reaction/hypersensitivity (drug fever)	6	0	1	0	0
Motor and Sensory Neuropathy	5	0	1	1	0
Muscle weakness, generalized, whole body	6	1	0	0	0
Diarrhea	5	2	0	0	0
Constipation	4	1	2	0	0
Anorexia	4	1	2	0	0
Nausea	2	3	2	0	0
Vomiting	3	3	1	0	0
Weight loss	4	1	2	0	0
Elevated Alkaline Phosphatase	5	2	0	0	0
Elevated iver Transaminases, AST	5	2	0	0	0
Elevated Liver Transaminases, ALT	2	5	0	0	0
Fever without neutropenia	4	3	0	0	0
Gastrointestinal - Other	4	1	2	0	0
Hiccoughs	6	1	0	0	0
Allergic rhinitis	5	1	1	0	0
Fatigue/Asthenia	0	1	6	0	0
Cytokine Release Syndrome	6	0	1	0	0
Diaphoresis	6	1	0	0	0
Dysarthria/Voice Changes	6	0	1	0	0
Dysgeusia/Taste Changes	4	1	2	0	0
Dyspepsia	6	0	1	0	0
Edema	6	1	0	0	0
Hyperbilirubinemia	6	1	0	0	0
Hypermagnesemia	4	3	0	0	0
Potassium, serum-high	6	1	0	0	0
Hypoalbuminemia	4	1	2	0	0
Hypocalcemia	5	1	1	0	0
Hypomagnesemia	5	1	1	0	0
Hyponatremia	4	3	0	0	0
Hypophosphatemia	5	1	0	1	0
Hypotension	5	0	2	0	0
Insomnia	6	1	0	0	0
Pain, all types	0	0	4	3	0
Rigors, Chills	5	1	1	0	0
Sino-nasal reactions	4	2	1	0	0
Skin breakdown/Decubitus Ulcer	6	0	1	0	0
Xerosis/ Dry Skin	3	3	1	0	0
Xerostomia/Dry Mouth	3	3	1	0	0

Correlative Studies of Serum Cytokines

Previously, a pre-treatment increase in multiple tumor-related cytokine and angiogenic growth factors was detected in patients with SCCHN.²⁶ Based on the rationale that this set of cytokines were co-regulated by NF-κB, the predictive value of coordinate changes in 3 or more of these cytokines was evaluated. Longitudinal increases in 3 or more of these factors was associated with decreased response and survival in patients with oropharyngeal SCCHN. Consistent with previous findings, increases in 3 or more cytokines occurred in 3 patients (2,3, 5, and 7) who developed PD, and increases in 2 or fewer cytokines, or decline, in 3 patients with CRs (1,4,6) (Fig. 2). Although based on fewer patients, there was evidence suggesting that those patients whose initial cytokine profile was generally associated with increasing values after starting treatment were more likely to have shorter progression free survival than those whose cytokine levels tended to decline with greater PFS (P=0.029 by exact two-tailed log-rank test). The patient showing the greatest increase in all 4 cytokines during cycle 1 was treated with the highest dose of bortezomib (#7, 1.3mg/m²), and developed PD in the neck by week 5 while on treatment.

Figure 2. Longitudinal changes in serum cytokine levels in seven patients.

Based on the rationale of co-regulation of 5 serum cytokines by NF-κB, the predictive value of coordinate changes in 3 or more of these cytokines was evaluated, as previously (23). Cytokine concentrations (pg/ml) are presented as mean ± standard deviation of replicates on a log scale vs. days since beginning treatment. Pre-Tx = pre-treatment. Longitudinal increase is slope of 3 or more cytokines previously associated with poor response and survival in patients with oropharyngeal carcinoma²³ was seen in patients 2, 3, 5, and 7, and associated with decreased progression free survival (P=0.029, log-rank test).

Correlative studies of markers of prosurvival signal, transcription factors and apoptosis

We examined the pharmacodynamic effects of bortezomib and cetuximab on EGFR, downstream signal, and apoptosis markers we previously validated for SCCHN in multiple studies.^{8, 10, 11, 15, 17-20} Only patient 7 consented to optional serial biopsies of SCCHN primary tumor, which were obtained pretreatment, on day 5 after induction with bortezomib 1.3 mg/m² (days 1, 4) and cetuximab 400 mg/m²(day 1), and on day 12, after combination with IMRT. Fig. 3A shows that by day 5, combination of bortezomib and cetuximab enhanced, rather than inhibited, phosphorylated and total EGFR, pERK1/2, and NF-κB p65 subunit. By day 12, with addition of IMRT, further enhancement of phosphorylated and total EGFR, pAKT, STAT3 and NF-κB p65 was observed. Increase in cleaved PARP as an indicator of cytotoxicity was only detected after initiation of IMRT (day 12).

Figure 3. Combined bortezomib, cetuximab and radiation enhances co-activation of EGFR and multiple prosurvival pathways in SCCHN tumor biopsies, along with clonogenic survival in line UMSCC-1.

A. Tumor biopsies were obtained from patient #7 before and on day 5 after initiating combined treatment with bortezomib and cetuximab, and on day 12 after addition of IMRT. Protein extracts from tumor specimens were subjected to SDS-PAGE and Western blots were performed for activated EGFR and signal phospho- and total proteins shown. Combined bortezomib and cetuximab treatment increased phosphorylation and total EGFR, and phosphorylation of downstream prosurvival signal kinases and transcription factors, including p-AKT, p-ERK1/2, p-STAT3 and p-NF-κB p65. B. Clonogenic survival assays. UMSCC-1 cells were treated with bortezomib, cetuximab, and RT alone or in combinations as indicated. Clonogenic assays were performed, and surviving fractions are presented. C. Western blots were performed for activated EGFR and signal phospho- and total proteins shown. Combined bortezomib and cetuximab treatment with RT resulted in stabilization of EGFR, and phosphorylated EGFR and downstream prosurvival signal kinases and transcription factors, including p-AKT, p-ERK1/2, p-STAT3.

Molecular effects in SCCHN in vitro

To further determine how these effects observed in tumor specimens were related to the activity of the individual or combination of agents, SCCHN cell line UMSCC-1 was treated as indicated, and effects were examined by clonogenic survival assay, and western blot for EGFR and downstream signaling components (Fig. 3B, C). Combination of C225 or bortezomib and radiation reduced clonogenic survival (Fig. 3B). However, combination of cetuximab and bortezomib when combined with radiation, reduced the overall effect of treatment to a level intermediate between that observed with either C225 or bortezomib with radiation, and the control (Fig. 3B). Reduction of survival was accompanied by reduction in EGFR and pEGFR (Fig. 3C). Inhibition of one or more downstream signal mediators including pAKT, pERK, and pSTAT3 was often observed with C225, or combination of C225 and radiation, but bortezomib attenuated these effects (Fig. 3C). These findings may explain the reduced efficacy of C225 and radiation when combined with bortezomib, which can inhibit proteasome activity, and possibly, C225-induced EGFR degradation.

Discussion

The combination of bortezomib, cetuximab, and IMRT was tolerated with supportive care, but resulted in a median PFS of only 4.8 months. These poor efficacy results included 5/6 previously untreated patients with HPV and/or p16 positive oropharyngeal carcinomas, which compared unfavorably to results of 17.1 months for oropharynx site tumors reported for cetuximab and radiotherapy.³ This group has been associated with favorable prognosis in additional studies, even though unfavorable characteristics (advanced stage and history of heavy smoking) which can influence outcome, were also present.²⁸ While it is possible these adverse risk factors contributed to the unexpectedly low response and early recurrence in the small cohort in the present study, translational studies provided additional evidence for an adverse interaction of the combination of bortezomib, cetuximab and radiation. Greater than expected EGFR and cell survival signaling, and angiogenesis factor expression by SCCHN was observed. Together, the clinical and molecular findings caution against further clinical investigation of this combination of agents.

The clinical results of this study were initially surprising after early preclinical and clinical studies provided evidence that combined treatment with either of these agents individually with radiation, or a combination of proteasome and EGFR inhibitors, potentiated cytotoxic activity.^{1, 2, 16, 17, 20-24} However, evidence emerging from one of our laboratories concurrent with this trial indicated that proteasome inhibitors could potentially antagonize chemotherapy or radiation induced EGFR degradation, and anti-proliferative and cytotoxic effects (M. Nyati, unpublished observations).²⁷ Consistent with this possibility, analysis of serial tumor biopsies from the patient who developed PD on-treatment revealed increased EGFR and prosurvival signaling instead of EGFR degradation and attenuation of prosurvival signaling previously reported with cetuximab or cetuximab and radiation.²⁷ Further studies in the UMSCC-1 cell line showed that combination of C225 or bortezomib with radiation reduced clonogenic survival consistent with previous preclinical studies (Fig. 3B), but combination of cetuximab and bortezomib with radiation, reduced the overall effect of treatment to a level intermediate between that observed with either drug with radiation, and the control (Fig. 3B). Bortezomib also attenuated the effects of cetuximab- and radiation-induced EGFR degradation, and inhibition of prosurvival signaling in UMSCC-1 (Fig. 3C).

Since recent evidence suggests EGFR is degraded by the ubiqutin-proteasome system,^30,31 it appears likely that proteasome inhibition by bortezomib could attenuate the cytotoxic effects of cetuximab and radiation by protecting EGFR from degradation. Further, recent reports demonstrate proteasome inhibitor-induced activation of EGFR as well as EGFR-independent mechanisms can induce MAPK, AKT and STAT3 prosurvival pathways, as observed here.^{9,19, 32-34} Additionally, while proteasome inhibitors radiosensitized cancer cells and smaller xenograft tumors in experimental models,^15-17 they may enhance radioprotection of SCC tumor cells under hypoxic conditions,³⁵ such as occur in large SCCHN in advanced stage patients. Cytokines and angiogenesis factors expressed by SCCHN in response to prosurvival²⁶ and hypoxia signals³⁶ were detected in serum of all 7 patients pretreatment. Concentrations of 3 or more serum cytokine and angiogenic growth factors previously shown to increase with poor response and survival in patients with oropharyngeal SCCHN,²⁶ increased in the 3 patients with early PD. We and others have shown that these cytokines may be produced in other patient tumors and cell lines by SCCHN epithelial and stromal cells.^37,38 We have further shown that cytokines such as IL-8 may be induced in SCCHN lines by bortezomib through activation of MAPK signaling and transcription factor AP-1. ³² Thus, proteasome inhibitor and EGFR induced expression of IL-8., VEGF and HGF could enhance angiogenesis, tumorigenesis and metastasis. ^{32, 37, 38}

Together, the stabilization or enhancement of EGFR-mediated survival signaling and angiogenesis factor expression may help further explain the suboptimal efficacy of the combination of these drugs with radiation. These observations suggest that cetuximab and radiation have multiple effects on cancer besides DNA repair, and that combination studies should be pursued with caution. Both drug-drug and drug-radiation interactions affecting diverse mechanisms may need to be considered when developing therapeutic regimens.

The first in human phase I study of bortezomib in combination with re-irradiation for recurrent SCCHN was also recently concluded at NIH. While bortezomib inhibited proteasome, NF-κB p65 subunit, and prosurvival genes,¹⁸ clinical activity of bortezomib plus reirradiation was limited.¹⁹ PRs were seen in 5/10 patients receiving lower doses and bortezomib treatment breaks, while PD occurred in patients receiving a continuous schedule or higher doses of bortezomib with re-irradiation. The limited clinical activity observed was also associated with lack of inhibition of EGFR-activated ERK or STAT3 pathways, as well as other non-canonical NF-κB/REL family members, which may also contribute to cell survival.¹⁹ A recent phase I study of bortezomib plus cetuximab in treatment refractory patients with tumors expressing EGFR yielded SD but no PRs or CRs in 5/6 with SCCHN or lung cancer.²⁴ Altogether, the results of these studies demonstrate that bortezomib in combination with cetuximab or re-irradiation results in incomplete clinical and molecular responses in SCCHN.

Recently completed phase II studies of bortezomib with other chemotherapies for recurrent SCCHN also showed evidence of limited combinatorial activity or possible chemoprotection.^39,40 One of these studies showed that the response rate was lower than expected for docetaxel alone, and PD was associated with an increased NF-κB and EGFR gene profile.³⁹ Another phase I trial evaluated bortezomib in combination with weekly cisplatin 30 mg/m² and RT for advanced SCCHN.⁴¹ Twenty-seven patients with previously untreated local-regionally advanced (10 patients) or recurrent/previously irradiated (17 patients) SCCHN were studied. Only 8 patients (30%) were without PD at a median 7.3 month follow-up. Interestingly, there is now also evidence that proteasome inhibition may antagonize chemotherapy-mediated EGFR degradation and cytotoxicity as well. Gemcitabine or cisplatin chemotherapy cytotoxicity was shown to involve ubiquitination and proteasome-dependent EGFR degradation.^{30, 42}

In conclusion, the present and other clinical and mechanistic studies suggest that bortezomib may have limited clinical efficacy, and in some instances, lower than expected activity due to antagonism, when combined with cetuximab and other cytotoxic therapies of known activity in SCCHN. Proteasome inhibitor-mediated activation of EGFR-dependent and independent MAPK, AKT or STAT3 prosurvival signaling may be countered by combination with ERK, JNK and AKT inhibitors. ^{31, 32} However, as learned here and other recent trials cited above, further study of proteasome inhibitors in combination with other targeted therapies should be considered only with caution after testing in appropriate non-HPV and HPV+ HNSCC xenograft models appropriate to the patient population to be studied. These results also provide several insights important in avoiding or reducing the impact of unfavorable outcomes in the future. Despite the inherent challenges and limitations in preclinical modeling of the combination and sequencing of multiple therapies to be used in clinical trials, accurate modeling is important to identify potential interactions and mechanisms that could result in unfavorable clinical outcomes. Close monitoring is important for early recognition of unfavorable outcomes for provision of additional therapy, early stoppage of the study, and reporting. Obtaining paired pre and on-treatment specimens for correlative studies can support the identification of possible underlying mechanisms.

Translational Relevance.

Epidermal growth factor receptor (EGFR) inhibitor cetuximab and radiotherapy is approved for squamous cell carcinoma of the head and neck (SCCHN), but the added benefit is limited to a subset of patients. EGFR inhibitors attenuate signaling via Mitogen Activated Protein Kinases (MAPKs) and Signal Transduction and Transcription Factor 3 (STAT3), while proteasome inhibitors block activation of Nuclear Factor-κB, another signal-activated transcription factor important in survival of SCCHN. Combined treatment with proteasome and EGFR inhibitors, or these agents individually with radiation demonstrated cytotoxic activity in preclinical and/or clinical studies. In this phase I trial, combining bortezomib with cetuximab and radiation therapy demonstrated unexpectedly short progression free survival that led to early study termination. There was evidence that bortezomib antagonized cetuximab- and radiation-induced degradation of EGFR, enhanced prosurvival signal pathway activation, and cell survival. Further clinical studies of proteasome inhibitors in combination with other therapies in SCCHN should be undertaken with caution.