Transforming growth factor-α: a surrogate endpoint biomarker?1

ORIGINAL SCIENTIFIC ARTICLES

Transforming Growth Factor-␣: A Surrogate Endpoint Biomarker? Samuel W Beenken, MD, FACS, Richard Hockett Jr, MD, William Grizzle, MD, PhD, Heidi L Weiss, PhD, Allan Pickens, MD, Marjorie Perloff, MD, Winfred F Malone, PhD, MPH, Kirby I Bland, MD, FACS Dysplastic oral leukoplakia (DOL) has been the index lesion in prevention trials for upper aerodigestive tract squamous cell carcinoma (SCC). Vitamin A derivatives, including 13-cis retinoic acid (13-CRA), have been used to treat DOL and to reduce the risk of subsequent SCC. Results from a trial of 13-CRA in patients with DOL are presented here. Transforming growth factor-␣ (TGF-␣) and the epidermal growth factor receptor messenger RNA (mRNA) expression were studied to validate their use as surrogate endpoint biomarkers in prevention trials for SCC. STUDY DESIGN: In a prospective, randomized, double-blind trial of 13-CRA in 28 patients with DOL, TGF-␣ and epidermal growth factor receptor mRNA expression were analyzed in sequential biopsy specimens of DOL and of adjacent normal-appearing mucosa, utilizing a quantitative, competitive, reverse transcriptase polymerase chain reaction and were compared using the Wilcoxon signed-rank test for paired comparisons. RESULTS: In biopsy specimens of DOL, TGF-␣ mRNA expression at baseline, but not baseline expression of epidermal growth factor receptor mRNA, was significantly elevated when compared with its expression in specimens from adjacent normal-appearing mucosa (p⫽0.003). In patients randomized to 13-CRA who had ⱖ50% clearance of DOL during treatment, significant modulation of TGF-␣ mRNA overexpression was seen after 6 months of treatment (p⫽0.016). TGF-␣ mRNA overexpression at baseline predicted a subsequent response to 13-CRA (p⫽ 0.066). CONCLUSIONS: The full extent of the association between TGF-␣ overexpression and the development of SCC is unknown. Evidence is presented in this article that TGF-␣ overexpression mediates the relationship between 13-CRA and DOL, but there is no direct evidence that it mediates the relationship between 13-CRA and the prevention of SCC. Determination of the extent to which TGF-␣ overexpression mediates this relationship and complete validation of TGF-␣’s role as a surrogate endpoint biomarker await the results of animal and human trials that utilize reduction in the incidence of SCC as their endpoint. (J Am Coll Surg 2002;195:149–158. © 2002 by the American College of Surgeons) BACKGROUND:

The goal of cancer prevention is to reduce the incidence of cancer in tissues at risk. To decrease the time and

expense involved in testing cancer prevention agents in randomized clinical trials, an intensive effort is being made to identify surrogate endpoint biomarkers (SEBs) for these trials. The upper aerodigestive tract provides a unique model system for study of the development of squamous cell carcinoma (SSC) and for the investigation of candidate SEBs. This model’s uniqueness derives from multistage tumorigenesis,1-4 field cancerization,5 and ease of access. Dysplastic oral leukoplakia (DOL) has been the index lesion in a series of prevention trials for upper aerodigestive tract SSC undertaken at our institution. DOL develops along epithelial surfaces that have been chronically exposed to carcinogens such as tobacco and alcohol.6 It is a marker of field cancerization and carries

No competing interests declared.

This work was supported in part by grant NCI 5 U01 CA68639-03 from the National Cancer Institute. Presented at the 6th International Symposium on Predictive Oncology and Intervention Strategies, February 2002, Paris, France. Received October 19, 2001; Revised March 8, 2002; Accepted March 11, 2002. From The University of Alabama at Birmingham, Department of Surgery (Beenken, Pickens, Bland) and Department of Pathology (Grizzle); the Biostatistics Unit of the Comprehensive Cancer Center, Birmingham, AL (Weiss); Eli Lilly and Co, Indianapolis, IN (Hockett); and the National Cancer Institute, Division of Chemoprevention, Bethesda, MD (Perloff, Malone). Correspondence address: Samuel W Beenken, MD, The University of Alabama at Birmingham, 1824 6th Ave South, 620 Wallace Tumor Institute, Birmingham, AL 35294-3300.

© 2002 by the American College of Surgeons Published by Elsevier Science Inc.

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Abbreviations and Acronyms

13-CRA DOL EGFr G3PDH NCI PCR QC-RT-PCR

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

SCC

⫽

SEB TGF-␣ UAB

⫽ ⫽ ⫽

13-cis retinoic acid dysplastic oral leukoplakia epidermal growth factor receptor glucose-3-phosphate dehydrogenase The National Cancer Institute polymerase chain reaction quantitative, competitive, reverse transcriptase polymerase chain reaction squamous cell carcinoma of the upper aerodigestive tract surrogate endpoint biomarker transforming growth factor-␣ The University of Alabama at Birmingham

a cancer risk of 50%.7-9 Since the early 1960s, vitamin A (retinol) and its derivatives (retinoids) have been used to treat DOL. Several studies documented initial resolution of DOL, but with frequent dose-related toxicity and with recurrence after cessation of treatment.10-13 Retinoic acid receptor-␤ has a role in mediating the cellular response to retinoids.14 The synthetic retinoid 13-cis retinoic acid (13-CRA) was effective in preventing a second SCC in patients with an earlier, successfully treated SCC.15,16 In an effort to define SEBs for SCC, we previously conducted a pilot clinical trial that utilized immunohistochemical techniques (Fig 1) to demonstrate: 1) baseline TGF-␣ and epidermal growth factor receptor

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(EGFr) protein overexpression in DOL when compared with their expression in adjacent normal-appearing mucosa and 2) modulation of TGF-␣ protein overexpression by 13-CRA.17,18 Subsequently, we have developed a high-throughput, quantitative, competitive, reverse transcriptase polymerase chain reaction (QT-RT-PCR) assay for the analysis of growth factors and receptors in biopsy specimens from the oral cavity. This assay uses internal competitors, has a large dynamic range, and is reproducible.19 This report presents results from a prospective, randomized, double-blind, cancer prevention trial that analyzed TGF-␣ and EGFr mRNA expression utilizing QC-RT-PCR in patients with DOL and no clinical or histologic evidence of SCC. METHODS Biopsy specimens

Six-millimeter punch biopsies were taken from areas of DOL and from adjacent normal-appearing mucosa before and after a 1-month observation period (pretreatment biopsies), and after 3, 6, and 12 months of treatment. Histologic examination of the pretreatment biopsies was performed to confirm the presence of DOL. Sequential biopsies were taken in close proximity to the preceding biopsy, but avoided its exact location. If resolution of DOL was seen, the subsequent leukoplakia biopsy was taken from an area of resolution. All patients signed an institutional review board-approved consent form indicating that biopsy specimens were for research

Figure 1. Immunohistochemical determination of TGF-␣ and EGFr protein expression. A) TGF-␣ expression. A transition from normal-appearing oral mucosa (left side) to DOL (right side) is shown. Note that an acellular keratin layer covers the DOL. There is a marked increase in the intensity of TGF-␣ staining in DOL. In the normal-appearing mucosa, staining is more intense in the basal layer and in the lower half of the mucosa, but is very low in the upper half of the mucosa. In DOL, the intensity of staining is uniform in all layers of the mucosa. B) EGFr expression. A transition from normal-appearing oral mucosa (right-side) to DOL (left-side) is shown. There is a marked increase in the intensity of EGFr staining in DOL, especially in the basal layer and in the lower half of the mucosa. DOL, dysplastic oral leukoplasia; EGFr, epidermal growth factor receptor; TGF-␣, transforming growth factor-␣.

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Figure 2. Schematic representation of the competitor pQPCR.GFO1.2. pQPCR.GFO1.2 was constructed by preparing oligonucleotides corresponding to the primers of TGF-␣, EGFr, and G3PDH mRNA and subcloning them into a pBluescript plasmid vector (Stratgene, La Jolla, CA) so that they flanked a “stuffer” segment (230-bp segment of murine genomic DNA). pQPCR.GFO1.2 contained a primer site for a T3 DNA-dependent RNA polymerase and also an oligonucleotide corresponding to the primer of ␣-actinin (␣-act), but ␣-actinin expression was not quantified in this trial. EGFr, epidermal growth factor receptor; G3PDH, glucose-3-phosphate dehydrogenase; TGF␣, transforming growth factor-␣.

purposes only. Additional biopsies were taken for diagnostic purposes if there was clinical evidence of disease progression that could require surgical therapy. The biopsy specimens taken for research purposes were trimmed to minimize the amount of subcutaneous tissue present and were then placed in OCT compound and snap-frozen in liquid nitrogen. Frozen specimens were subsequently removed from OCT with a razor blade and placed in 300 ␮L guanidine isothiocyanate solution (4 M GITC, 15mM Na citrate, 0.3% SLS, pH 7.0, 0.1 M 2-mercaptoethanol, 10 ␮g/mL MS-2 phage RNA) and homogenized for 30 seconds with a tissue homogenizer (Fisher Scientific Co, Suwanee, GA). Baseline TGF-␣ and EGFr mRNA expression

Baseline TGF-␣ and EGFr mRNA expression was defined as the average of the mRNA expression values determined from biopsy specimens taken before and after a 1-month observation period (pretreatment biopsies). In brief, a competitor RNA “minigene” was developed that expressed single-stranded RNA competitors that were spiked at different concentrations into tissue extracts from biopsy specimens before RNA extraction and cDNA synthesis. Construction of the competitor RNA minigene, pQPCR.GFO1.2 (Fig 2), and quantification

of TGF-␣, EGFr, and glucose-3-phosphate dehydrogenase (G3PDH) mRNA expression were performed as previously described.19,20 Ten microliters of each tissue extract in guanidine isothiocyanate solution was utilized per titration point. Reverse transcription of mRNA into cDNA was performed. Polymerase chain reaction (PCR) was performed in 20-␮L reactions with 2 ␮L cDNA in 0.5-mL Eppendorf tubes for 30 to 35 cycles of 94°C for 30 seconds, 55°C for 1 minute, and 72°C for 30 seconds. Final MgCl2 concentrations were 2.6mM. PCR primers were: TGF-␣ upstream 5⬘-TGGACACACGTGGAACC; TGF-␣ downstream 5⬘-CCTCCTTCTGTGACTGG; EGFr upstream 5⬘-AGGAGGAGGCTTGCTGG; EGFr downstream 5⬘-GGCTGGAATCCGAGTTA; G3PDH upstream 5⬘-TCCTGCACCACCAACTG; G3PDH downstream 5-GCCTGCTTCACCACCTT. After PCR amplification, an ELISA assay was used to quantify mRNA expression. The stuffer segments of the single-stranded RNA competitors allowed for their detection. Hybridization oligonucleotides were: TGF-␣ 5⬘-GAGGCTCTAACACTGCTCAGGAG AC;

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Table 1. Clinical Response of Dysplastic Oral Leukoplakia to 13-CRA Treatment Response

Complete response

Partial response

Stable disease Progression

Description

95% or greater decrease in the product of the two greatest diameters of a lesion or in the sum of products when there is more than one lesion 50% or greater decrease in the product of the two greatest diameters of a lesion or in the sum of products when there is more than one lesion Less than a partial response, but without progression 25% or greater increase in the product of the two greatest diameters of a lesion or in the sum of products when there is more than one lesion or the development of new lesions

EGFr 5⬘-GCCGGATCGGTACTGTATCAAGTCA; G3PDH 5⬘-CCTGACCTGCCGTCTAGAAAAAC CT; Stuffer 5⬘-GATCCTCAAGGGTCGAGACTGTCAT AC.

Paired comparisons of mRNA expression in DOL and in adjacent normal-appearing mucosa were made for each patient. The Wilcoxon signed-rank test for paired comparisons was used to determine important differences. Modulation of TGF-␣ and EGFr mRNA expression

Following the 1-month observation period, patients were randomized to a 3-month course of 13-CRA at 1.0 mg/kg/day (“high-dose”) or to placebo. Response to treatment (Table 1) was monitored at monthly clinic visits. After 3 months, the treatment status of patients who had not responded to treatment (stable disease or disease progression) was determined. Patients found to be taking placebo were immediately started on a 3-month course of high-dose 13-CRA. All patients completing the prescribed 3-month course were continued on 13-CRA for an additional nine months at the reduced dose of 0.25mg/kg/day (“low-dose”) to minimize longterm toxicity. To facilitate the accurate comparison of TGF-␣ and EGFr mRNA expression before, during, and after 13CRA treatment, all mRNA extracts from a given patient underwent QC-RT-PCR analysis as a batch. For each patient, baseline mRNA expression was compared with mRNA expression at 3, 6, and 12 months (“paired com-

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parisons”) to evaluate modulation of mRNA expression by 13-CRA. The Wilcoxon signed-rank test for paired comparisons was used to determine if notable modulation occurred. RESULTS Twenty-eight patients were enrolled in the study, 14 patients each in the placebo and 13-CRA arms. The mean age of the patients was 69.4 years. Sixty-four percent of the patients were female and 96% were Caucasian. Clinical response

Six patients came off the study early because they did not complete the initial 3 months of treatment (three in the placebo arm and three in the 13-CRA arm). To facilitate description of the clinical response to treatment in the remaining 22 patients, three patient groups were defined (Table 2). Group I

Four patients randomized to placebo had a partial response and subsequently began low-dose 13-CRA. Group II

Seven patients randomized to placebo showed no response and were started on high-dose 13-CRA, with six of these patients completing the prescribed 3-month course—one patient had a complete response (16.7%), two patients had a partial response (33.3%), and three patients had stable DOL (50.0%). Group III

All 11 patients randomized to 13-CRA completed the prescribed 3-month course of high-dose treatment. Four patients had a complete response (36.4%), three patients had a partial response (27.3%), and four patients had stable DOL (36.4%). In summary, 17 of the 18 patients treated with highdose 13-CRA completed the prescribed 3-month course; 10 patients had at least a partial response (58.8%) and 5 had a complete response (29.4%). During the subsequent 9 months of low-dose 13-CRA, three additional patients had a complete response, but five patients, including one of those who had had a complete response after 3 months of high-dose 13-CRA, showed progression. There was no major difference seen in the severity of dysplasia in the biopsy specimens of patients with DOL who responded to 13-CRA when compared with those of patients who did not respond. No correlation between 13-CRA treatment and the development

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Table 2. Clinical Response of Dysplastic Oral Leukoplakia to Placebo or 13-CRA Patients Group*

No.

I

C†

020 005 010 024 027‡ 003 008 015 018 002 025 019 001 016 021 004 006 026‡ 013 007 012 028‡

II

III

Response to high-dose 13-CRA

Response to placebo P

SD

Pro

C

P

SD

Pro

X X X X

Response to low-dose 13-CRA C

P

SD

Pro

Off study X X X Off study

X X X X X X X

X X X

X X X X

X X

X

X

X X

X X X X

X X X

X X X

X X X

X X X X

Off study X X X

*See text: Results, Clinical Response. † C, complete response; P, partial response; SD, stable disease; Pro, progression (see Table 1). ‡ mRNA expression not analyzed. 13-CRA, 13-cis retinoic acid.

of SCC could be demonstrated during the short timecourse of this trial, but one patient (004, Table 2), who had a partial response while receiving high-dose 13CRA, developed SCC after commencing low-dose 13CRA. Baseline TGF-␣ and EGFr mRNA expression

Baseline TGF-␣ mRNA expression was determined in biopsy specimens of 25 patients with DOL and in 23 biopsy specimens of the adjacent normal-appearing mu-

cosa, which permitted 23 paired comparisons. Baseline expression of TGF-␣ mRNA in biopsy specimens of DOL, but not expression of EGFr mRNA, was significantly increased when compared with its expression in specimens of adjacent normal-appearing mucosa (p⫽ 0.003, Table 3). Modulation of TGF-␣ and EGFr mRNA expression

Modulation of TGF-␣ and EGFR mRNA expression during treatment with 13-CRA was analyzed in two pa-

Table 3. Paired Comparison of Baseline* TGF-␣† and EGFr mRNA Expression Dysplastic oral leukoplakia

TGF-␣ EGFr

Normal-appearing mucosa

n

Mean ⴞ SE

Median

n

Mean ⴞ SE

Median

25 25

4,938.1 ⫾ 666.4 85,877.7 ⫾ 27,643.1

4,025.0 50,998.2

23 23

2,663.3 ⫾ 413.7 83,059.2 ⫾ 21,441.3

2,212.9 46,984.3

‡

§

*Average of two pretreatment mRNA expression values. † TGF-␣: transforming growth factor-␣; EGFr: epidermal growth factor receptor. ‡ mRNA analysis was not performed for three patients (see Table 2). § mRNA analysis of normal-mucosa specimens from two patients was not performed. 㛳 p ⫽ 0.003 (Wilcoxon signed-rank test for paired comparisons).

Difference n

Mean ⴞ SE

23 ⫺1,940.9 ⫾ 704.5 23 ⫺6,454.3 ⫾ 29,020.1

Median

⫺1,271.0㛳 ⫺3,142.6

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in response to 13-CRA was associated with TGF-␣ mRNA overexpression at baseline (p⫽0.066). Patients who were randomized to placebo and subsequently started high-dose 13-CRA (Table 2, group II) were seen to have low-level TGF-␣ mRNA expression in DOL at baseline and showed no modulation of TGF-␣ mRNA expression during treatment (data not shown). 13-CRA did not alter either TGF-␣ or EGFr mRNA expression in biopsy specimens of adjacent normal-appearing mucosa. Placebo did not alter either TGF-␣ or EGFr mRNA expression in biopsy specimens of DOL or in specimens of adjacent normal-appearing mucosa. Figure 3. Modulation of TGF-␣ mRNA expression in dysplastic oral leukoplakia by 13-CRA. Three months of high-dose 13-CRA (1.0 mg/kg/day) was followed by 9 months of low-dose treatment (0.25 mg/kg/day). X axis: 13-CRA treatment times; Y axis: mean TGF-␣ mRNA expression by QC-RT-PCR. Standard error bars are shown. There was significant modulation of TGF-␣ mRNA expression at 6 months (p⫽0.016, Wilcoxon signed-rank test for paired comparisons). 13-CRA, 13-cis retinoic acid; QC-RT-PCR, quantitative, competitive, reverse transcriptase polymerase chain reaction; TGF-␣, transforming growth factor-␣.

tient groups. 1) Six of seven patients who were randomized to placebo, but showed no response, completed the subsequent 3-month course of high-dose 13-CRA and 9-month course of low-dose 13-CRA (Table 2, group II). No modulation of TGF-␣ or EGFr mRNA expression was seen in this group. 2) Ten of 11 patients who were randomized to 13-CRA completed the prescribed 3-month course of high-dose treatment and the subsequent 9-month course of low-dose treatment (Table 2, group III). Modulation of TGF-␣ mRNA overexpression at 6 months of treatment was seen in those patients who had ⱖ50% clearance of DOL (p⫽0.016) (Fig 3, Table 4). Significant modulation of TGF-␣ mRNA expression

DISCUSSION Progress in cancer prevention is being seriously slowed by a lack of SEBs that can be substituted for the costly “true endpoint” of cancer incidence reduction in prevention trials. SEBs are tissue, cellular, and molecular changes that correlate with the later development of cancer. SEBs are needed to replace the use of cancer incidence reduction as the endpoint for cancer prevention trials, thereby shortening the duration of the trial, reducing the sample size required, and providing early or more specific indicators of cancer risk and the efficacy of intervention. Another important but separate goal of biomarker research is to identify biomarkers that will predict with great accuracy those patients who will respond to a particular therapy (predictive biomarkers). In the trial reported here, baseline TGF-␣ mRNA overexpression in DOL was predictive of significant modulation of TGF-␣ mRNA expression (p⫽0.066). In this context, TGF-␣ overexpression might define a subset of patients at risk for SCC who have acquired unique genetic abnormalities or carry predisposing genetic alterations or both. It is not known whether TGF-␣ mRNA overex-

Table 4. 13-CRA Modulation of TGF-␣ and EGFr mRNA Expression in Dysplastic Oral Leukoplakia* TGF-␣ Treatment, mo

0 3 6 12

EGFr

Mean ⴞ SE

Median (n)

Mean ⴞ SE

Median (n)

7,926.3 ⫾ 2,042.2 7,400.0 ⫾ 3,328.9 2,534.0 ⫾ 724.7 1,407.2 ⫾ 351.9

5,929.0 (9) 4,301.0 (8) 2,246.5‡ (8) 1,438.0 (5)

69,463.1 ⫾ 13,032.0 131,938.4 ⫾ 67,996.0 29,693.3 ⫾ 7,121.2 34,348.8 ⫾ 9,923.0

62,979.0 (9) 56,096.0 (8) 21,966.0 (8) 24,838.0 (5)

†

*Analysis of mRNA expression in biopsy specimens of patients who were randomized to 13-CRA and had ⱖ50% clearance of dysplastic oral leukoplakia during treatment. † Fewer specimens were available in the later months because not all patients had completed treatment at the time of mRNA analysis. ‡ p ⫽ 0.016, Wilcoxon signed-rank test for paired comparisons. 13-CRA, 13-cis retinoic acid; EGFr, epidermal growth factor receptor; TGF-␣, transforming growth factor-␣.

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Figure 4. Receptor tyrosine kinase activation. The binding of a ligand, such as TGF-␣, to a receptor, such as EGFr, leads to receptor dimerization, activation of its kinase activity, and phosphorylation of tyrosine residues outside the catalytic domain that recruit a number of signaling proteins. A cascade of signaling events is initiated, leading to changes in the expression of genes involved in the control of cell-cycle progression, cell differentiation, and cell death. [Adapted with permission from: Lamorte L, Park M. The receptor tyrosine kinases. In Brodt P, ed. Surg Oncol Clin North Am 2001; 10(2):271–288.] EGFr, epidermal growth factor receptor; TGF-␣, transforming growth factor-␣.

pression in DOL is predictive of an increased incidence of SCC or of a quicker progression to SCC. Although the validity of predictive biomarkers can often be readily demonstrated, the validity of a SEB can only be determined when the effect of a prevention agent on the SEB is concordant with its effect on the true endpoint of cancer incidence reduction.21 The complexity of the interactions among growth factors, cytokines, and hormones, and their receptors makes validation of TGF-␣ in this regard very difficult. In addition, although considerable evidence supports the use of clinical/histologic lesions as SEBs, such as severe cervical intraepithelial neoplasia for cervical cancer and adenomatous polyps for colon carcinoma, DOL is not a good candidate SEB for SCC because many SCCs do not develop in DOL, which is primarily a marker for field cancerization. Control of cell proliferation and differentiation is me-

diated in part by the interaction of growth factors such as TGF-␣, cytokines, and hormones and their receptors, such as EGFr.22 Receptor activation triggers a cascade of intracellular signals that result in the activation and repression of various genes involved in the control of cell proliferation, cell differentiation, and cell death (Fig 4). Receptors with tyrosine kinase activity, such as EGFr, have an extracellular ligand-binding domain and a cytosolic domain containing a catalytic kinase domain.23 In the absence of a ligand such as TGF-␣, receptors are catalytically inactive. Ligand binding promotes receptor dimerization, which induces a conformational change in the receptor and activates its kinase.24 Once activated, the receptor tyrosine kinase phosphorylates tyrosine residues outside the kinase domain, which then serve as binding sites for proteins involved in signal transduction.25 Through a series of specific protein–protein interactions, the activated receptor assembles a complex of

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Table 5. Solid Tumors Showing Alterations in Receptors with Tyrosine Kinase Activity Receptor amplification

Receptor

Tumor

Reference

EGFr

Breast SCC Breast SCC Breast Gastric Gastric Melanoma Breast Colon Thyroid Thyroid GIST Hepatocellular MEN IIA MEN IIB

43 44 45,46 46 47,48 48 49 50 51 52 53,54 53,55 56 57 58,59 60,61

FGF HER-2/neu

Autocrine loops* Chromosome translocations

Point Mutations

Met FGF Met TRK-A RET c-Kit Met RET

*Coexpression of a receptor with tyrosine kinase activity and its growth factor. FGF, fibroblast growth factor; GIST, gastrointestinal stromal tumor; MEN (IIA, IIB), multiple endocrine neoplasia (IIA, IIB); SCC, squamous cell carcinoma of the upper aerodigestive tract.

proteins involved in signal transduction that transmits a signal from the cell surface to the nucleus. Alterations in this process, such as receptor amplification, autocrine loops, chromosome translocations, and point mutations can contribute to the development of cancer (Table 5). Given the complexity of the interactions between growth factors, cytokines, and hormones and their receptors, validation of TGF-␣ as a SEB necessitates answering three questions: 1) Is TGF-␣ mRNA overexpression associated with SCC? 2) Does treatment with 13-CRA modulate TGF-␣ overexpression? 3) Does TGF-␣ overexpression mediate the relationship between 13-CRA and prevention of SCC?

traoral location of DOL or the degree of dysplasia present.18 In the trial presented in this article, we used QT-RT-PCR to demonstrate baseline TGF-␣ mRNA overexpression in DOL when compared with its baseline expression in adjacent normal-appearing mucosa (Table 3). The mechanisms underlying TGF-␣ mRNA and protein overexpression in DOL and SCC might include alterations in genetic controls secondary to carcinogenic insult or autocrine effects, or both.37 In SCC cell lines, steady-state TGF-␣ mRNA overexpression was shown to result primarily from an increase in the rate of gene transcription and was not accounted for by increased gene copy number or alterations in message stability.38

Is TGF-␣ overexpression associated with SCC?

Does treatment with 13-CRA modulate TGF-␣ overexpression?

The evidence that TGF-␣ participates in the development of SCC comes from transfection studies using mouse mammary epithelial cell lines26 and from analyses of mRNA and protein from a variety of cultured human carcinoma cell lines,27-31 including SCC.33,34 In humans, TGF-␣ mRNA overexpression has been shown in SCC.35,36 In a pilot cancer prevention trial, we demonstrated TGF-␣ protein overexpression at baseline in DOL, a premalignant lesion for SCC, when compared with its baseline expression in adjacent normalappearing mucosa.17,18 That trial also demonstrated excellent correlation of TGF-␣ expression between two pretreatment biopsy specimens, irregardless of the in-

In our earlier pilot trial, we demonstrated modulation of TGF-␣ protein overexpression by 13-CRA,17,18 and in the randomized, placebo-controlled trial presented in this article, we have demonstrated that 13-CRA modulates TGF-␣ mRNA overexpression in DOL (Fig. 3, Table 4). Retinoids were also shown to modulate TGF-␣ mRNA overexpression in SCC cell lines, where Northern analysis showed a mean reduction of 35.4% in 9 of 10 cell lines after exposure to all-transretinoic acid.38 Although the mechanisms underlying the effect of retinoids on TGF-␣ protein and mRNA overexpression are not fully understood, retinoids are known to regulate the

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development, growth, and differentiation of cells.39-41 They appear to exert most of their effect by altering gene expression.42 In SCC cell lines, nuclear run-on assays suggested that the effect of all-transretinoic acid on TGF-␣ mRNA overexpression occurred primarily via decreased gene transcription.38 Exposure to alltransretinoic acid normalized TGF-␣ mRNA expression in these cell lines. Baseline EGFr protein overexpression was shown in DOL17,18 and EGFr expression can be modulated by retinoids.38 Although we did not demonstrate baseline EGFr mRNA overexpression in DOL or modulation of EGFr mRNA expression by 13-CRA in this trial, trends in those directions were seen (Tables 3 and 4). The small number of specimens analyzed might have prevented demonstration of EGFr overexpression and modulation. Does TGF-␣ overexpression mediate the relationship between 13-CRA and prevention of SCC?

Evidence that TGF-␣ mRNA overexpression mediates the relationship between 13-CRA and DOL includes: 1) Expression of TGF-␣ mRNA at baseline in biopsy specimens of DOL is significantly increased when compared with its expression in adjacent normal-appearing mucosa (p⫽0.003); 2) in patients randomized to 13-CRA who had ⱖ50% clearance of DOL during treatment, modulation of TGF-␣ mRNA overexpression is seen after 6 months of treatment (p⫽0.016); and 3) TGF-␣ overexpression at baseline in DOL predicts a subsequent response to 13-CRA. But there is no direct evidence that TGF-␣ overexpression mediates the relationship between 13-CRA and the prevention of SCC. Although a randomized, placebo-controlled trial showed that 13CRA can prevent a second SCC, in patients with an earlier, successfully treated SCC,15,16 TGF-␣ expression was not analyzed in that study. Determination of the extent to which TGF-␣ overexpression mediates the relation between 13-CRA and the prevention of SCC and validation of TGF-␣ as an SEB awaits the results of animal and human trials that specifically analyze the relationship between modulation of TGF-␣ overexpression and reduction in the incidence of SCC. Author Contributions

Study conception and design: Beenken, Hockett, Grizzle, Weiss, Perloff, Malone Acquisition of data: Beenken, Hockett, Grizzle, Pickens

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