tucci, A. Franchi (Institute of Anatomic Pathol- ogy), University of Florence, Italy. .... PH, Stanley MA, Ozanne B, et al. Associa- tion of CDKN2A/p16INK4A with ...
BRIEF COMMUNICATION Cumulative Prognostic Value of p16/CDKN2 and p53 Oncoprotein Expression in Premalignant Laryngeal Lesions Oreste Gallo, Marco Santucci, Alessandro Franchi* Cancers of the head and neck, particularly of the larynx, are often preceded by epithelial lesions showing different histologic alterations (1-3). As it has been hypothesized for other cancers (4), each step in the development of head and neck cancers may be the result of the accumulation of genetic damage in oncogenes and tumor-suppressor genes. To assess the potential value of the p16 and p53 oncoproteins as markers of malignant progression, we undertook a retrospective immunohistochemical analysis using a series of biopsy specimens from patients with precancerous conditions of the larynx. Twenty-six consecutive patients with precancerous laryngeal lesions who were treated by microlaryngoscopy and vocal cord stripping with biopsy, but who had not received any further treatment except close follow-up, and experienced a malignant progression of the disease were selected. Patients were excluded from our analysis based on the following criteria: 1) initial biopsy performed at another hospital (three case subjects); 2) history of previous lung or head and neck carcinomas (three case subjects); and 3) diagnosis of invasive carcinoma less than 1 year after the first biopsy, presumably because of a sampling error made at the time of initial biopsy (four case subjects). Thus, 16 patients formed the basis of this brief communication. There were 13 men and three women; the median age at initial diagnosis was 61.5 years (range, 37-73
years). Each original lesion was reviewed and graded as hyperplasia (eight case subjects), mild dysplasia (four case subjects), moderate dysplasia (two case subjects), and severe dysplasia or carcinoma in situ (two case subjects), according to the criteria of Friedmann (5). The control group consisted of 32 patients with laryngeal lesions and risk factor exposure comparable to those of the study population and who had no progression of their disease during a period comparable to the follow-up of the first group (6.7 years; range, 1.8-11 years), after initial microlaryngoscopy and vocal cord stripping. The control subjects were also selected on the basis of tobacco use after initial surgery (in the study group, four of 12 smokers did not quit smoking after initial diagnosis). The median age (61.5 years; range, 3671 years) and male-to-female ratio (4.3/ 1) of the control subjects were comparable to the study population. The immunohistochemical studies were performed on formalin-fixed, paraffin-embedded sections and involved use of the avidin–biotin–complex technique (Dakopatts, High Wycombe, U.K.). To identify alterations in p16 expression, we used an anti-p16 polyclonal antibody (Pharmingen, San Diego, CA) and for p53 detection the monoclonal antibody DO7 (Dako Co., Carpinteria, CA) was used. p53 immunostaining required microwave pretreatment of dewaxed and rehydrated sections. Negative controls were performed by substitution of the primary antibody with nonimmune rabbit or mouse serum. Positive controls for p16 immunostaining consisted of paraffin sections of normal laryngeal mucosa and of cutaneous melanocytic nevi. To score the p16 staining pattern, we used the criteria proposed by Geradts and Wilson (6). Only cells with evidence of nuclear staining were considered positive. If all cells within a lesion exhibited nuclear staining for p16, the pattern was considered normal; any reduction of the number of stained cells in the presence of positive staining of stromal cells was considered an abnormal pattern. The immunostaining was regarded as inconclusive when stromal cells lacked nuclear reactivity. p53 immunostaining was scored according to the semiquantitative criteria proposed by Barbatis et al. (7):
Journal of the National Cancer Institute, Vol. 89, No. 15, August 6, 1997
− 4 negative staining; + 4 less than 10% of positive cells; ++ 4 10%-50% of positive cells; and +++ 4 more than 50% of positive cells. Lesions scored as ++/+++ were considered as showing overexpression of the oncoprotein. Statistical tests were performed with the use of Stata software (Stata Corp., College Station, TX). The relationship between clinical and pathologic variables was assayed by the two-tailed Fisher’s exact test or by the two-tailed Pearson’s chi-squared method. P values less than .05 were considered significant. As previously reported (6), immunostaining for p16 varied in intensity but generally displayed nuclear reactivity in both normal cells and in pathologic conditions (Fig. 1, A). In three (6.3%) of 48 samples of preneoplastic laryngeal lesions, the p16 status could not be determined, since there was complete absence of nuclear reactivity in the epithelial lamina and surrounding stromal tissue, possibly because of damage to the p16 protein during fixation and tissue processing (6). These three case subjects included one with severe dysplasia coupled with malignant evolution and two with premalignant lesions without tumor progression during follow-up (one with severe dysplasia and one with carcinoma in situ). All of these patients with unevaluable disease were excluded from our analysis. Thus, we compared results from 15 patients who had progressing premalignant laryngeal diseases with those from 30 patients who had premalignant disease only. The analysis of p16 expression according to the histopathologic grading of the premalignant lesions is shown in Table 1. The comparative analysis of p16 expression in biopsy samples from patients with premalignant lesions whose disease did and did not progress to laryngeal cancer did not show any
*Affiliations of authors: O. Gallo (Institute of Otolaryngology-Head & Neck Surgery), M. Santucci, A. Franchi (Institute of Anatomic Pathology), University of Florence, Italy. Correspondence to: Oreste Gallo, M.D., Institute of Otolaryngology-Head & Neck Surgery, University of Florence-Policlinico di Careggi, V.le G.B. Morgagni 85, 50134 Firenze, Italy. See ‘‘Note’’ following ‘‘References.’’ © Oxford University Press
BRIEF COMMUNICATION 1161
Fig. 1. Aberrant expression of p16 (A) and p53 (B) in a case of moderate dysplasia of the larynx. Only few cells of the epithelial lamina display nuclear positivity for p16, while mucosal inflammatory cells and fibroblasts show intense nuclear immunostaining (A). Expression of p53 is diffuse and limited to the epithelial lamina (B). This patient developed a squamous cell carcinoma of the larynx 4 years later.
significant statistical difference (60% versus 46.7%; P 4 .59). All progressing premalignant lesions with altered p16 expression (nine of 15 4 60%) showed p16 alteration in the corresponding metachronous carcinoma. Twenty-six (57.8%) of 45 precancerous lesions were positive for p53 (Fig. 1, B). In all but one of the p53-positive carcinomas that have developed in patients with previous p53-positive premalignant lesions and in five (45.5%) of 11 squamous cell carcinomas that had developed in patients with p53 overexpression, we observed an increased expression in comparison with the corresponding premalignant lesions. Comparative analysis of p53 expression among transformed or nontransformed precancerous lesions failed to prove that
p53 was a prognostic factor (five of 15 4 33.3% of evolved lesions versus 18 of 30 4 60% of nonevolved lesions; P 4 .75). p53 oncoprotein expression tended to be higher in lesions from subjects who smoked; however, this trend was not statistically significant (P 4 .09). Simultaneous p53 and p16 alterations had a prognostic relevance in tumor progression, being present in 60% of progressing and in only 23.3% of nonprogressing precancerous lesions (P 4 .03). Our results indicate that precancerous laryngeal lesions can have altered expression of products of more than one tumor-suppressor gene (e.g., p16 and p53). These findings, together with the known association between aberrant protein expression and damage of both
genes (7-10), strongly support the genetic progression model proposed by Califano et al. (11). These authors suggested that inactivation of the p16 and p53 tumor-suppressor genes is the earliest detectable event in the multistep head and neck carcinogenesis. The finding that a significant percentage of precancerous laryngeal lesions with multiple phenotypic alterations is more prone to progress to cancer supports this model of progression, suggesting that laryngeal cancer is caused by a series of genetic events, each potentially leading to a clonal outgrowth of cells through selective growth advantages; however, it is the accumulation and not necessarily the order of such alterations that determines the progression. Accordingly, we report that the simultaneous alterations of the expression of the two tumorsuppressor genes have a higher prognostic value in cancer risk assessment for premalignant laryngeal lesions than that of each gene per se. Similarly, Mao et al. (12) have reported the cumulative prognostic value of multiple genetic alterations (loss of heterozygosity) at 9p21 and 3p14) in cancer risk assessment of premalignant oral lesions, suggesting the possible early involvement of both the p16 and the FHIT gene, a putative tumor-suppressor gene described as altered in head and neck carcinomas (13). In conclusion, the immunohistochemical study of multiple biomarkers could help to identify patients in whom precancerous laryngeal lesions are prone to progress to invasive cancer. These patients at high risk could be selected for chemoprevention, which seems to reduce the progression of premalignant disease (14,15), or for endoscopic screening protocols.
Table 1. Overall p16 and p53 expression in premalignant and malignant laryngeal lesions, according to smoking and drinking habits No. of patients (%)
No. of smokers (%) No. of drinkers (%)
p16 absent or low expression p53 overexpression Alteration of both proteins
Hyperplasia (n 4 24)
Mild dysplasia (n 4 12)
Moderate dysplasia (n 4 6)
Severe dysplasia/ carcinoma in situ (n 4 3)
8 (33.3)
7 (58.3)
5 (83.3)
3 (100)
11 (73.3)
.02
18 (54.5)
6 (50)
5 (83.3)
2 (66.7)
11 (73.3)
.26
4 (33.3)
5 (83.3)
2 (66.7)
9 (60)
.01
13 (54) 5 (21)
Squamous cell carcinoma (n 4 15)
P
Heavy/ moderate smokers* (n 4 33)
Nonsmokers (n 4 12)
P
Drinkers† (n 4 24)
Nondrinkers† (n 4 21)
P
6 (50)
.946
13 (54.2)
11 (52.3)
.92
22 (66.7)
4 (33.3)
.09
16 (66.7)
10 (47.6)
.32
12 (36.4)
4 (33.3)
.48
10 (41.7)
6 (28.6)
.78
*Heavy smokers: ù30 pack-years. Moderate smokers:
