Phase II randomized trial of radiation therapy, cetuximab, and pemetrexed with or without bevacizumab in patients with locally advanced head and neck cancer.

BACKGROUND: We previously reported the safety of concurrent cetuximab, an antibody against epidermal growth factor receptor (EGFR), pemetrexed, and radiation therapy (RT) in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN). In this non-comparative phase II randomized trial, we evaluated this non-platinum combination with or without bevacizumab, an inhibitor of vascular endothelial growth factor (VEGF). PATIENTS AND METHODS: Patients with previously untreated stage III-IVB SCCHN were randomized to receive: conventionally fractionated radiation (70 Gy), concurrent cetuximab, and concurrent pemetrexed (arm A); or the identical regimen plus concurrent bevacizumab followed by bevacizumab maintenance for 24 weeks (arm B). The primary end point was 2-year progression-free survival (PFS), with each arm compared with historical control. Exploratory analyses included the relationship of established prognostic factors to PFS and quality of life (QoL). RESULTS: Seventy-eight patients were randomized: 66 oropharynx (42 HPV-positive, 15 HPV-negative, 9 unknown) and 12 larynx; 38 (49%) had heavy tobacco exposure. Two-year PFS was 79% [90% confidence interval (CI) 0.69-0.92; P < 0.0001] for arm A and 75% (90% CI 0.64-0.88; P < 0.0001) for arm B, both higher than historical control. No differences in PFS were observed for stage, tobacco history, HPV status, or type of center (community versus academic). A significantly increased rate of hemorrhage occurred in arm B. SCCHN-specific QoL declined acutely, with marked improvement but residual symptom burden 1 year post-treatment. CONCLUSIONS: RT with a concurrent non-platinum regimen of cetuximab and pemetrexed is feasible in academic and community settings, demonstrating expected toxicities and promising efficacy. Adding bevacizumab increased toxicity without apparent improvement in efficacy, countering the hypothesis that dual EGFR-VEGF targeting would overcome radiation resistance, and enhance clinical benefit. Further development of cetuximab, pemetrexed, and RT will require additional prospective study in defined, high-risk populations where treatment intensification is justified.

Collagen type XI α1 facilitates head and neck squamous cell cancer growth and invasion.

BACKGROUND: Although it is well established that the extracellular matrix affects tumour progression, not much is known about the various components and their effect on head and neck squamous cell carcinoma (HNSCC) progression. Levels of collagen type XI α1 (colXIα1), a minor fibrillar collagen, have been shown to be increased in tumour compared with normal tissue in several cancers, including colorectal, breast, and non-small cell lung cancer. Currently, the functional significance of colXIα1 is not understood. METHODS: We examined the expression levels of colXIα1 mRNA and elucidated the functional role of colXIα1 in HNSCC. Cell proliferation, invasion, and migration were examined with and without colXIα1 knockdown with siRNA in HNSCC cells. RESULTS: Our data demonstrate that colXIα1 expression is increased in tumour samples compared with levels in normal adjacent tissue in 16/23 HNSCC patients. In addition, colα11 is increased in HNSCC cell lines compared with normal immortalised epithelial cells and is increased in tumour-derived fibroblasts compared with normal fibroblasts. Using an siRNA approach, we demonstrate that colXIα1 contributes to proliferation, migration, and invasion of HNSCC. CONCLUSION: Our cumulative findings suggest that colXIα1 contributes to HNSCC tumorigenesis and may serve as a potential therapeutic target.

Cetuximab and bevacizumab: preclinical data and phase II trial in recurrent or metastatic squamous cell carcinoma of the head and neck.

BACKGROUND: We evaluated combined targeting with cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, and bevacizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody, in squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: The combination was studied in human endothelial cells and head and neck and lung cancer xenograft model systems. Patients with recurrent or metastatic SCCHN were treated with weekly cetuximab and bevacizumab, 15 mg/kg on day 1 given intravenously every 21 days, until disease progression. Analysis of tumor biomarkers and related serum cytokines was performed. RESULTS: Cetuximab plus bevacizumab enhanced growth inhibition both in vitro and in vivo, and resulted in potent reduction in tumor vascularization. In the clinical trial, 46 eligible patients were enrolled. The objective response rate was 16% and the disease control rate 73%. The median progression-free survival and overall survival were 2.8 and 7.5 months, respectively. Grade 3-4 adverse events were expected and occurred in less than 10% of patients. transforming growth factor alpha, placenta-derived growth factor, EGFR, VEGFR2 increased and VEGF decreased after treatment but did not correlate with treatment efficacy. CONCLUSIONS: Cetuximab and bevacizumab are supported by preclinical observations and are well tolerated and active in previously treated patients with SCCHN.

Phase I trial of pemetrexed in combination with cetuximab and concurrent radiotherapy in patients with head and neck cancer.

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/m(2) (level -1, 200 mg/m(2); level +1, 500 mg/m(2)) 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/m(2) in cohort A and 350 mg/m(2) 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/m(2) to cetuximab and radiotherapy is recommended for further study in not previously irradiated patients.

Response assessment by combined PET-CT scan versus CT scan alone using RECIST in patients with locally advanced head and neck cancer treated with chemoradiotherapy.

PURPOSE: RECIST have limitations when applied to potentially curable locally advanced squamous cell carcinoma of the head and neck (SCCHN). [¹8F]fluorodeoxyglucose-positron emission tomography (PET) scan may be useful in assessing treatment response and predicting patient outcome. PATIENTS AND METHODS: We studied patients with previously untreated stages III-IVb SCCHN treated with primary concurrent chemoradiotherapy on five prospective clinical trials. Response was assessed by clinical exam, computed tomography (CT), and PET portions of combined PET-CT scan ∼8 weeks after completion of chemoradiotherapy. RESULTS: Fifty-three patients were analyzed. Complete response (CR) was demonstrated in 42 patients (79%) by clinical exam, 15 (28%) by CT, and 27 (51%) by PET. CR as assessed by PET, but not as assessed by clinical exam or CT using RECIST, correlated significantly with progression-free status (PFS) (P < 0.0001). The 2-year PFS for patients with CR and without CR by PET was 93% and 48%, respectively (P = 0.0002). CONCLUSIONS: A negative PET scan on combined PET-CT after chemoradiotherapy is a powerful predictor of outcome in patients receiving curative chemoradiotherapy for SCCHN. PET-CT is indicated for response evaluation in this setting to improve the accuracy of post-treatment assessment by CT.

Leveraging Genomics for Head and Neck Cancer Treatment.

The genomic landscape of head and neck squamous cell carcinoma (HNSCC) has been recently elucidated. Key epigenetic and genetic characteristics of this cancer have been reported and substantiated in multiple data sets, including those distinctive to the growing subset of human papilloma virus (HPV)-associated tumors. This increased understanding of the molecular underpinnings of HNSCC has not resulted in new approaches to treatment. Three Food and Drug Administration-approved molecular targeting agents are currently available to treat recurrent/metastatic disease, but these have exhibited efficacy only in subsets of HNSCC patients, and thus surgery, chemotherapy, and/or radiation remain as standard approaches. The lack of predictive biomarkers to any therapy represents an obstacle to achieving the promise of precision medicine. This review aims to familiarize the reader with current insights into the HNSCC genomic landscape, discuss the currently approved and promising molecular targeting agents under exploration in laboratories and clinics, and consider precision medicine approaches to HNSCC.

Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor- mediated cell growth In vitro.

Stimulation of epidermal growth factor receptor (EGFR) by ligand(s) leads to activation of signaling molecules including Stat1 and Stat3, two members of the signal transducers and activators of transcription (STAT) protein family. Activation of Stat1 and Stat3 was constitutive in transformed squamous epithelial cells, which produce elevated levels of TGF-alpha, and was enhanced by the addition of exogenous TGF-alpha. Targeting of Stat3 using antisense oligonucleotides directed against the translation initiation site, resulted in significant growth inhibition. In addition, cells stably transfected with dominant negative mutant Stat3 constructs failed to proliferate in vitro. In contrast, targeting of Stat1 using either antisense or dominant-negative strategies had no effect on cell growth. Thus, TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat3 but not Stat1.

IL6 is associated with response to dasatinib and cetuximab: Phase II clinical trial with mechanistic correlatives in cetuximab-resistant head and neck cancer.

OBJECTIVE: Src family kinase (SFK) activation circumvents epidermal growth factor receptor (EGFR) targeting in head and neck squamous cell carcinoma (HNSCC); dual SFK-EGFR targeting could overcome cetuximab resistance. PATIENTS AND METHODS: We conducted a Simon two-stage, phase II trial of the SFK inhibitor, dasatinib, and cetuximab in biomarker-unselected patients with cetuximab-resistant, recurrent/metastatic HNSCC. Pre- and post-treatment serum levels of interleukin-6 (IL6) were measured by ELISA. HNSCC cell lines were assessed for viability and effects of IL6 modulation following dasatinib-cetuximab treatment. RESULTS: In the first stage, 13 patients were evaluable for response: 7 had progressive and 6 had stable disease (SD). Enrollment was halted for futility, and biomarker analysis initiated. Low serum IL6 levels were associated with SD (raw p=0.028, adjusted p=0.14) and improved overall survival (p=0.010). The IL6 classifier was validated in a separate trial of the same combination, but was unable to segregate survival risk in a clinical trial of cetuximab and bevacizumab suggesting serum IL6 may be specific for the dasatinib-cetuximab combination. Enhanced in vitro HNSCC cell death was observed with dasatinib-cetuximab versus single agent treatment; addition of IL6-containing media abrogated this effect. CONCLUSION: Clinical benefit and overall survival from the dasatinib-cetuximab combination were improved among patients with low serum IL6. Preclinical studies support IL6 as a modifier of dasatinib-cetuximab response. In the setting of clinical cetuximab resistance, serum IL6 is a candidate predictive marker specific for combined dasatinib-cetuximab. The trial was modified and redesigned as a biomarker-enriched Phase II study enrolling patients with undetectable IL6.

Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter.

BACKGROUND: Squamous cell carcinomas of the head and neck (SCCHN), unlike normal mucosal squamous epithelial cells, overexpress epidermal growth factor receptor (EGFR) messenger RNA and protein. EGFR protein is required to sustain the proliferation of SCCHN cells in vitro. To determine whether EGFR expression contributes to tumor growth, we investigated the effect of suppressing EGFR expression in tumor xenografts through in situ expression of antisense oligonucleotides. METHODS: Intratumoral cationic liposome-mediated gene transfer was used to deliver plasmids capable of expressing sense or antisense EGFR sequences into human head and neck tumors, which were grown as subcutaneous xenografts in nude mice. The oligonucleotides were expressed under the control of the U6 RNA promoter. RESULTS: Direct inoculation of the EGFR antisense (but not the corresponding sense) plasmid construct into established SCCHN xenografts resulted in inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of apoptosis (programmed cell death). Sustained antitumor effects were observed for up to 2 weeks after the treatments were discontinued. CONCLUSION: These results suggest that interference with EGFR expression, using an antisense-based gene therapy approach, may be an effective means of treating EGFR-overexpressing tumors, including SCCHN.

Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer.

The squamous mucosa of patients who develop head and neck cancer is "condemned" or predisposed to disregulated growth as reflected by the high incidence of synchronous and metachronous primary tumors. We hypothesized that transformed and nontransformed mucosa from head and neck cancer patients would produce increased levels of transforming growth factor alpha (TGF-alpha) and its cell surface receptor, the epidermal growth factor receptor (EGFR), thereby contributing to this predisposition. Using molecular biological techniques, we examined the incidence and mechanism of TGF-alpha and EGFR overproduction in tumors and histologically normal mucosa excised from patients with squamous cell carcinoma of the head and neck (SCCHN) to test this hypothetical mechanism of field cancerization. Northern blot hybridization was used to evaluate the frequency of increased TGF-alpha and EGFR mRNA production in tissue excised from 24 patients with SCCHN and 10 cell lines compared with 7 control patients without cancer or a history of alcohol and tobacco use. Southern blot hybridization was used to examine for gene amplification. In patients with SCCHN, TGF-alpha mRNA was elevated by a mean of 5-fold in 95% of histologically "normal" mucosa samples (P = 0.001) and by a mean of 5-fold in 87.5% of tumors (P = 0.0001) while EGFR mRNA was elevated by a mean of 29-fold in 91% of histologically normal mucosa specimens (P = 0.0005) and by a mean of 69-fold in 92% of tumors (P = 0.0005), compared with mRNA levels in control normal mucosa. In 10 SCCHN cell lines, TGF-alpha mRNA was increased by a mean of 16-fold and EGFR mRNA levels were increased by a mean of 77-fold. Increased production of TGF-alpha and EGFR mRNA in the histologically normal mucosa of patients at risk for a primary or secondary head and neck cancer may serve both as a marker for malignant transformation and as a target for preventive therapies.

Abrogation of transforming growth factor-alpha/epidermal growth factor receptor autocrine signaling by an RXR-selective retinoid (LGD1069, Targretin) in head and neck cancer cell lines.

Clinical studies have demonstrated that retinoids, including retinol (Vitamin A) and its synthetic derivatives, can eradicate leukoplakia and suppress the formation of squamous cell carcinoma of the head and neck (SCCHN). Nonselective retinoids have been shown to abrogate transcriptional activation of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR), which characterize SCCHN. LGD1069 (Targretin) is a potent RXR-selective retinoic acid agonist with a reduced toxicity profile compared with other nonselective retinoids. We examined the effect of LGD1069 (10 microm) on cellular proliferation and expression of putative intermediate biomarker genes including TGF-alpha, EGFR, and RAR-beta in seven SCCHN cell lines. A quantitative reverse transcription-PCR assay using a novel "primer dropping" method was used to determine expression levels of EGFR, TGF-alpha, and RAR-beta before and after treatment with LGD1069 (10 microM). SCCHN proliferation was reduced by a mean of 50% at 4 days in seven SCCHN cell lines after LGD1069 treatment (P < or = 0.05). EGFR expression levels were decreased by a mean of 58.4% (P = 0.007), TGF-alpha levels were decreased by a mean of 28.8% (P = 0.01), and RAR-beta levels were increased by a mean of 60% (P = 0.03). TGF-alpha stimulation of EGFR is associated with constitutive signal transducer and activator of transcription 3 (Stat3) activation in SCCHN. Abrogation of constitutive Stat3 activation was seen with LGD1069 treatment. These results suggest that an RXR-selective retinoic acid decreases SCCHN proliferation in part by interfering with TGF-alpha/EGFR autocrine signaling.

Nucleic acid targeting: towards personalized therapy for head and neck cancer.

In light of a detailed characterization of genetic aberrations in cancer, nucleic acid targeting represents an attractive therapeutic approach with significant translational potential. Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer deaths worldwide with stagnant 5-year survival rates. Advances in conventional treatment have done little to improve survival and combined chemoradiation is associated with significant adverse effects. Recent reports have characterized the genetic alterations in HNSCC and demonstrated that mutations confer resistance to conventional and molecular targeted therapies. The ability to use specific nucleic acid sequences to inhibit cancer-associated genes including non-druggable targets facilitates personalized medicine approaches with less adverse effects. Additionally, advances in drug delivery mechanisms have increased the transfection efficiency aiding in greater therapeutic responses. Given these advances, the stage has been set to translate the information garnered from genomic studies into personalized treatment strategies. Genes involved in the tumor protein 53 and epidermal growth factor receptor pathways have been extensively investigated and many promising preclinical studies have shown tumor inhibition through genetic modulation. We, and others, have demonstrated that targeting oncogene expression with gene therapy approaches is feasible in patients. Other methods such as RNA interference have proven to be effective and are potential candidates for clinical studies. This review summarizes the major advances in sequence-specific gene modulation in the preclinical setting and in clinical trials in head and neck cancer patients.

Frequent promoter hypermethylation of PTPRT increases STAT3 activation and sensitivity to STAT3 inhibition in head and neck cancer.

Signal transducer and activator of transcription 3 (STAT3) overactivation is a common event in many cancers, including head and neck squamous cell carcinoma (HNSCC), where STAT3 represents a promising therapeutic target. HNSCC is not characterized by frequent kinase mutations, in contrast to some malignancies where mutational activation of kinases upstream of STAT3 is common. Instead, STAT3 may be activated by loss-of-function of negative regulators of STAT3, including by promoter hypermethylation of PTPRT. Here we first analyzed The Cancer Genome Atlas data and determined that the PTPRT promoter is frequently hypermethylated in several cancers, including HNSCC (60.1% of tumors analyzed) in association with downregulation of PTPRT mRNA expression and upregulation of pSTAT3 expression. These findings were confirmed in an independent cohort of HNSCC tumors by methylation-specific PCR and immunohistochemistry. We demonstrate that PTPRT promoter methylation and gene silencing is reversible in HNSCC cells, leading to PTPRT-specific downregulation of pSTAT3 expression. We further show that PTPRT promoter methylation is significantly associated with sensitivity to STAT3 inhibition in HNSCC cells, suggesting that PTPRT promoter methylation may serve as a predictive biomarker for responsiveness to STAT3 inhibitors in clinical development.

STAT signaling in head and neck cancer.

The upper aerodigestive tract is predisposed to the formation of multiple primary tumors due to field cancerization. TGF-alpha/EGFR autocrine signaling appears to play an important role in squamous cell carcinoma of the head and neck (SCCHN) and upregulation of TGF-alpha and EGFR is an early event in SCCHN carcinogenesis. STAT proteins, including Stat3, are activated by TGF-alpha and EGFR and strategies that downmodulate TGF-alpha or EGFR inhibit SCCHN cell proliferation and abrogate Stat3 activation. Targeting Stat3 leads to SCCHN growth inhibition, increases apoptosis and a downmodulation of Bcl-xL expression in head and neck tumors. These studies support the role of Stat3 as an oncogene, which is activated early in SCCHN carcinogenesis, and efforts to understand EGFR-mediated Stat3 signaling could facilitate novel strategies that will interfere with this growth promoting pathway. Oncogene (2000).

Differential activation of apoptosis regulatory pathways during monocytic vs granulocytic differentiation: a requirement for Bcl-X(L)and XIAP in the prolonged survival of monocytic cells.

Neutrophils and monocytes/macrophages are derived from common progenitors, but exhibit markedly different lifespans. Differentiated neutrophils are short-lived and die rapidly by apoptosis, while monocytic cells are longer-lived. In this report we used the HL-60 cell line as a model system to identify differences in apoptotic pathways which might account for the differing lifespans of granulocytic vs monocytic cells. We observed that induction of granulocytic differentiation by retinoic acid led to robust activation of the executioner protease caspase-3, and early onset of apoptosis. By contrast, caspase-3 was not appreciably activated during phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation, and apoptosis was delayed in these cells. Since the activation of caspase-3 is inhibited by members of the inhibitor of apoptosis (IAP) and Bcl-2 protein families, we investigated the expression of anti-apoptotic members of these families. Induction of monocytic differentiation led to marked upregulation of the IAP protein XIAP, as well as the Bcl-2 family member Bcl-X(L). During granulocytic differentiation the levels of XIAP progressively declined, while Bcl-X(L) levels remained unchanged. A different IAP protein, survivin, was downregulated during differentiation along either lineage, as was expression of Bcl-2. The upregulation of Bcl-X(L) during monocytic differentiation coincided with phosphorylation/activation of STAT3, a known activator of bcl-X gene transcription. Moreover, Bcl-X(L) upregulation was dependent on MEK/ERK signaling. Upregulation of XIAP proceeded in a MEK/ERK-independent fashion. Treatment with antisense Bcl-X(L) or XIAP oligonucleotides resulted in significant loss of viability in cells differentiating along the monocytic lineage. Together, these findings indicate that the levels of XIAP and Bcl-X(L) are regulated by distinct pathways during monocytic differentiation, and that upregulation of these proteins contributes to the increased longevity of cells in the monocytic lineage.

Human leukocyte antigen class I allelic and haplotype loss in squamous cell carcinoma of the head and neck: clinical and immunogenetic consequences.

The expression of human leukocyte antigen (HLA) class I molecules on the cell surface is necessary for the presentation of peptide antigens to cytotoxic CD8+ T lymphocytes of the immune system. Down-regulation of HLA class I gene expression has been implicated in tumorigenesis, including squamous cell carcinoma of the head and neck (SCCHN). Loss of MHC class I antigens may be one mechanism by which tumor cells escape immune detection. We performed prospective immunostaining of 26 primary SCCHN tumors and samples of normal mucosa harvested several centimeters away from the primary tumor, using a large panel of antibodies directed against allele-specific as well as monomorphic determinants of HLA class I molecules. Loss of expression of HLA class I proteins in the tumor was found in 50% (13 of 26) of primary tumors and was highly correlated with HLA loss in the corresponding normal mucosa (P < 0.0001). Further analysis demonstrated that the loss of HLA class I expression in the tumor was significantly associated with regional lymph node metastases (nodal stage; P = 0.0388), and that the number of HLA class I alleles lost in the normal mucosa was associated with subsequent development of a new primary aerodigestive tract cancer (P = 0.042). A patient with two metachronous cancers available for analysis had no evidence of HLA loss in the first tumor, demonstrated allelic loss in the second cancer, and subsequently died of disease. These results suggest that the loss of expression of HLA class I alleles may have prognostic implications.

Determination of intermediate biomarker expression levels by quantitative reverse transcription-polymerase chain reaction in oral mucosa of cancer patients treated with liarozole.

Liarozole is a 1-substituted imidazole derivative that inhibits cytochrome P450 activity and increases endogenous plasma concentrations of retinoid acid (RA). We have previously demonstrated that RA down-modulates transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) levels in head and neck squamous cell carcinoma by decreasing the transcription rate of these two genes. Previous reports suggest that RA receptor (RAR)-beta levels are down-modulated in head and neck cancer and are restored by RA therapy. Cellular RA-binding protein (CRABP)-II is up-regulated by RA and appears to modulate intracellular RA metabolism. In conjunction with a Phase I clinical trial, total intact RNA was extracted from oral cavity mucosa biopsied from 17 patients with advanced malignancies, before and after treatment with a 4-week course of liarozole. To analyze these limited quantities of total RNA (as little as 0.6 microg/sample), a quantitative reverse transcription-PCR assay was developed using delayed dropping of the 5' beta-actin primer to amplify the highly abundant beta-actin gene as an internal control. We used this method to determine the expression levels of TGF-alpha, EGFR, RAR-beta, and CRABP-II before and after treatment. There was a trend toward elevation of RAR-beta levels in oral mucosa after liarozole therapy (P = 0.107), whereas TGF-alpha, EGFR, and CRABP-II were not modulated by systemic liarozole treatment. These results suggest that liarozole may up-regulate RAR-beta in tissues from cancer patients and that expression levels of potential intermediate biomarkers may be determined in small tissue biopsies using a quantitative reverse transcription-PCR assay.

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium.

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.

Normalization of EGFR mRNA levels following restoration of wild-type p53 in a head and neck squamous cell carcinoma cell line.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by mutation of the p53 tumor suppressor gene and increased expression of the epidermal growth factor receptor (EGFR). To determine the potential link between p53 and EGFR expression, we examined the effect of overexpressing wild-type p53 on EGFR mRNA levels in HNSCC. In these studies, a temperature-sensitive (ts) p53 mutant was transfected into an HNSCC line which contained a deletion of one allele of the p53 gene and a mutation of the remaining allele. Following selection, six clones were isolated, characterized by Southern blot analysis, and stable expression of mutant p53 protein was confirmed by immunoblotting of clones grown at the mutant temperature. Total RNA was isolated from transfectants grown at both wild-type or mutant temperatures followed by Northern blot analysis to determine levels of EGFR mRNA expression at the two p53 conformations. Clones grown at the wild-type temperature (32.5 degreesC) demonstrated a 69% 13% decrease in EGFR mRNA compared with the same cells grown at the mutant temperature (39.5 degreesC; p=0.006) indicating that restoration of wild-type p53 reduces EGFR mRNA levels in this HNSCC cell line. These findings suggest that abnormalities in p53 may contribute to activation of EGFR gene transcription.

Synergistic enhancement by interleukin-1 alpha of cisplatin-mediated antitumor activity in RIF-1 tumor-bearing C3H/HeJ mice.

Administration of interleukin-1 alpha (IL-1 alpha) plus certain cytotoxic drugs causes substantially greater clonogenic tumor-cell kill and tumor-regrowth delay than does treatment with either agent alone. IL-1 alpha itself has little effect on tumor growth despite its ability to induce acute hemorrhagic necrosis, restrict tumor blood flow, and cause microvascular injury in a variety of murine model systems. To investigate further IL-1 alpha's ability to enhance the antitumor activity of cytotoxic drugs, we initiated studies to examine the effect of IL-1 alpha on cisplatin (cDDP)-mediated cytotoxicity using the RIF-1 tumor system. The antitumor activity of IL-1 alpha and cDDP was quantitated through standard clonogenic tumor-cell survival assays, a tumor hemorrhagic necrosis assay and tumor-regrowth delay studies, with the interaction between IL-1 alpha and cDDP being analyzed through median dose-effect. In vitro, IL-1 alpha had no enhancing effect on the cDDP-mediated tumor-cell kill. For examination of the in vivo efficacy of this regimen, RIF-1 tumor-bearing C3H/HeJ mice (14 days postimplantation) were treated concurrently with single i.p. injections of IL-1 alpha and/or cDDP at various doses. The increased clonogenic tumor-cell kill obtained with IL-1 alpha/cDDP was dose-dependent, with significant enhancement by IL-1 alpha being observed (P < 0.001), even at the lowest doses tested (2 mg/kg and 6 micrograms/kg for cDDP and IL-1 alpha, respectively), but it did not correlate with an increase in tumor hemorrhage. Using median dose-effect analysis, this interaction was determined to be strongly synergistic. When treated animals were monitored for long-term antitumor effects, combinations with IL-1 alpha significantly increased the tumor-regrowth delay and decreased the fractional tumor volume (P < 0.001). These results demonstrate that IL-1 alpha synergistically enhances cDDP mediated in vivo antitumor activity and suggest that the combination of IL-1 alpha and cDDP may have potential therapeutic application in the design of effective treatment modalities for cancer.