17

p53, Rb, and Cycli Verrucous Carcino

Irma B. Gimenez-Conti, D.D.s.’ Ana M. Collet, D.D.s.’-~ Hector Lanfranchi, D.D.S? Maria E. Itoiz, D.D.s.~ Mario Luna, M.D!

Shi-Xue Hu, M.D? William F. Benedict, M.D.~ Claudio J. Conti, D.v.M., P h n ’

Hong-Ji XU, M.D., Ph.D.’

I Science Park-Research Division, The Univer- sity of Texas M. D. Anderson Cancer Center, Smithville, Texas.

Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas.

Oral Pathology Department, Faculty of Odon- tology, University of Buenos Aires, Buenos Ai- res, Argentina.

Department of Molecular Oncology, The Uni- versity of Texas M.D. Anderson Cancer Center, Houston, Texas.

Department of Hematology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas.

Supported by Grant CTR 53322R1 from the Council for Tobacco Research and CA54672 from the National Cancer institute.

The authors thank the histology laboratory for their technical assistance, the art department for their services, and Carrie McKinley for her secretarial help.

Address for reprints: Claudio J. Conti, D.V.M., Ph.D., M. D. Anderson Cancer Center, Science Park-Research Division, P.O. Box 389, Smiths- ville, TX 78957.

Received March 19, 1996; accepted April 2, 1996.

n D1 Expression in Human Oral mas

BACKGROUND. The verrucous carcinoma (VC), a tumor with low grade malignancy, appears to be associated with tobacco and human papillomavirus. The pathobiol- ogy of these tumors has not been extensively studied, and molecular genetic alter- ations have not been reported. In this study we investigated by immunohistocheni- istry the expression of p53, Rb, and cyclin D1 in a series of well-defined oral VC. Changes in the expression of these genes have been commonly reported in a variety of human tumors. METHODS. We studied 29 cases of VC, fixed in formalin and embedded in paraffin. Immunohistochemistry was carried out using the avidin-biotin immunoperoxidase technique. Polyclonal antibody CM-1 was used for p53, a rabbit polyclonal human RB antibody, Rb-WL-1 antibody for Rb and a rabbit polyclonal human cyclin D antibody for cyclin D1. RESULTS. Positive p53 expression (protein accumulation) was detected in 15 of the 29 VC analyzed. In some cases, p53-positive areas were small foci but in most of the cases extensive positive areas were observed. None of the cases studied showed alterations of Rb protein. The expression of cyclin D1 was determined in 18 cases of VC. Positive nuclear immunostaining was seen in 11 cases. CONCLUSIONS. p53 protein accumulation is frequently observed in these tumors suggesting possible mutations of this gene in VC. Overexpression of cyclin D1 but no alterations of Rb staining were also observed in this low grade tumor suggesting that Rb may be functionally inactivated by overexpression of cyclin D1 or HPV infection. Cancer 1996; 7817-23. 0 1996 American Cancer Society.

KEYWORDS: p53, Rb, Cyclin D1, head and neck, verrucous carcinomas.

errucous carcinoma (VC) was defined by Ackerman in 1948 as a V distinct pathology of the oral cavity.’ It appears to be associated with tobacco and human papillomavirus (HPV) as the two most im- portant rick

Clinically, histologically, and biologically VC is an entity with low grade malignancy, slow growth, and no metastatic potential. The typi- cal oral lesion differs from the frequent squamous cell carcinoma (SCC) in that it is generally slow growing and chiefly exophytic, pro- ducing a warty clinical appearance. Endophytic growth results in local invasion, with little risk of

Microscopically, VC is characterized by extreme thickening of the epithelium with broad bulbous rete ridges pushing into the connec- tive tissue along a broad, even front. The basement membrane is intact, differentiation is of a high order, and the epithelium shows little mitotic activity, pleomorphism, or hyperchromatism. However, more aggressive types may occur which exhibit nuclear atypias and even in situ carcinoma areas. Inadequate treatment of these cases may lead to transformation into SCC. Characteristically, cleftlike spaces lined by a thick layer of parakeratin extend from the surface

0 1996 American Cancer Society

18 CANCER July 1,1996 / Volume 78 / Number 1

deeply into the lesion. Parakeratin plugging also ex- tends into the epithelium.* The diagnosis of VC is based on a review of both clinical and pathologic fea- tures, including growth rate, spread, and gross and microscopic appearance.

Molecular genetic alterations have not been stud- ied in VC and the only important finding regarding the pathogenesis of this lesion is the presence of HPV in a large number of cases.'-.' However, HPV may be present only as a passenger virus since the well differ- entiated epithelium of VC is a favorite host cell envi- ronment for the replication of the papilloma viruses.

In this study, we investigated possible alterations of the p53, Rb genes, and cyclin D1 in a series of well- defined oral VC. p53 and Rb are well-characterized tu- mor suppressor genes. p53 encodes a nuclear phospho- protein that regulates proliferation and apoptosis and seems to play a role in preventing DNA damage. Muta- tions of the p53 gene have been shown to be the most frequently occurring genetic abnormalities in human malignancies." In the head and neck region, ,953 gene mutations have been identified as occurring in SCC cell linesl I I I ? as well as in primary tumor^.'^^'“ Also, overex- pression of p53 protein, as detected by immunohisto- chemistry technique, has been reported in 54 to 67% of oral cancers. 15-" Another suppressor gene that has been studied in head and neck or other tumors is the retino- blastoma gene. This gene is a prototypical tumor sup- pressor gene initially identified in patients with the he- reditary form of retinoblast~ma.'~.'~ The Rb gene en- codes a 110 to 116 kilodalton (kD) nuclear protein with cell cycle-specific changes in phosphorylation and DNA- binding proper tie^.'^-^^ The loss of Rb function impli- cated in the development of retinoblastoma has also been identified as playing a key role in the pathogenesis by initiation and/or progression of some of the most common human malignancies, including carcinomas of the lung, breast, prostate, and bladder, and sarcomas of the soft tissue and b ~ n e . ~ ~ - ~ "

Cyclins are another family of proteins that have been recognized in playing a role in the development of neo- plasia. :"." These proteins are the regulatory domain of cyclin dependent kinases (CDK) and are believed to regulate transit through the cell cycle. Particularly cyclin D1, expressed in early G1, and shown to be amplified, rearranged, or overexpressed in a wide variety of tumors including head and neck carcinoma^.^^-^^

We report here that p53 protein accumulation and cyclin D1 overexpression are frequently observed in this low grade tumor but alterations in Rb protein were not observed.

MATERIALS AND METHODS Specimens Twenty-nine paraffin embedded tissue samples from oral biopsy specimens diagnosed as VC were retrieved

from the files of the pathology department in the School of Dentistry at the University of Buenos Aires. Of the 28 patients, only 3 were confirmed smokers and 17 were nonsmokers. This data was not available for the rest of the patients. All specimens had been rou- tinely fixed in formalin and embedded in paraffin. Se- rial 5 pm sections were cut from each sample. One section from each sample was stained with hematoxy- lin and eosin (H & E); the adjacent sections on poly- L-lysine coated slides were immunostained.

lmrnunohistochemical Studies Immunostaining for p53 and cyclin D1 was performed as previously described.37 After deparaffinization and rehydration of these tissue samples, endogenous per- oxidases were blocked. An antigen-retrieval tech- nique3' (BioGenix Labs, San Ramon, CA) to unmask antigen was used prior to half hour incubation with polyclonal antibody CM-1 (1:35 working dilution) for p533g9-41 (Signet Labs, Inc., Dedham, MA), and rabbit polyclonal human cyclin D (5 pglmL) (UBI, Lake Placid, NY) .42 A biotinylated antirabbit immunoglobu- lin (Ig) (Vector Laboratories, Burlingame, CA) was then applied for 30 minutes before the use of the avidin- biotin complex (ABC) (Vector Laboratories). The speci- mens were then incubated with a solution of diamino- benzidine tetrahydrochloride and hydrogen peroxide for 7 to 10 minutes. A dark brown precipitate within the nucleus confirmed the presence of the p53 or cyclin D1 protein.

Paraffin sections of oral SCC and chemically in- duced tumors4' were used as positive controls for p53 and cyclin D1, respectively. Human oral mucosa as well as representative lesions stained with normal se- rum instead of the primary antibody were used as negative controls.

The immunostain for R b was performed as pre- viously described.43 The Rb-WL-1 antibody in a final concentration of 2 pg/mL was used followed by the ABC technique. Sections were briefly counterstained with Mayer's hematoxylin & eosin before mounting.

RESULTS The histopathologic diagnosis of 29 cases of VC of the oral mucosa was confirmed by clinical followup of the patients. These surgical specimens included a wide range of grades going from less aggressive lesions to more aggressive and invasive behavior. In general, the microscopic feature was a well-differentiated, hyper- plastic stratified squamous epithelium exhibiting little or no increased mitotic activity or cellular atypia. In some cases the tumor showed a heavy inflammatory cell reaction in the adjacent connective tissue.

Of the 28 patients, only 3 were confirmed smokers, thus it was not possible to make any correlation be-

p53, Rb, Cyclin in Verrucous CarcinomalGimenez-Conti et al. 19

TABLE I Expression of p53, and Cyclin D1 in Human Oral Verrucous Carcinomas by Immunohistochemistry

Immunohistochemistry

Sample # Agelsex P53 Rb Cyclin D1

1 NIA tt t t 2 64IF t t 3 741M t -

6 NIA t t 7 67IM t -

9 54IM ttt t NIA 10 741F tt t t 11 60IM - NIA -

12 74IF ttt t NIA 13 751F tttt t t 17 75IM - NIA NIA 18a 52IM tttt NIA NIA 20 651F tt NIA NIA 21 46IM t NIA N /A 22 NIA - NIA NIA 23 63IF tttt NIA NIA 24a 521M tttt NIA NIA 26 34IM tttt N /A NIA 27 NIA - t NIA 28 801F t t 29 801F t t 30 67IF t t 31 64IM t -

32 831M tt t -

33 381M t -

34 75IF tt t t 35 781F t t 36 72IF ttt t -

37 38IF tt t t Total: 29

-: no immunostained nuceli; t: few, scattered (<lo%); tt: small foci (<lo%); ttt: large area (10- 50%); t t t -: all (50- 100%); NIA: no available data. " Both samples are from the same patient. Sample I8 is a surgical biopsy made one year after sample 24. Samples 28 and 29 are from the same patient.

- - - -

- - - -

-

-

tween tobacco history and histologic or molecular phenotype of the lesion.

Immunohistochemical studies to detect accumu- lation of p53 were carried out using the polyclonal antibody CM- 1. We previously demonstrated that this antibody, used under the same conditions of this study, does not detect normal levels of p53 and that it only labels cells that have accumulated p53 protein as a result of mutations or other physiologic or patho- logic condition^.^'

Elevated p53 expression was detected in 15 of the 29 VC analyzed (Table 1). In all cases the staining was restricted to the nuclei. In Cases 1, 10, and 21, the p53 positive areas were small foci (Fig. lA), but in other cases (Fig. lB) , extensive positive areas were observed. No positive staining was observed in the stromal or inflammatory cells. Immunostaining was also carried

out in the adjacent normal mucosa of all the cases in which was available and in 5 normal mucosa from noncancer patients. All those specimens were p53 neg- ative. (Fig. 1G). From the 14 VC p53 negative cases, 8 were well-differentiated lesions and the rest only presented some areas with dysplasia.

Immunohistochemical detection of Rb was car- ried out using the Rb-Wl-1 polyclonal antibody, as described by Xu et al.43 None of the cases studied showed alterations of the Rb protein. Positively stained nuclei were observed in the basal and su- prabasal layers of the epithelia and in the VCs. Posi- tive staining of stromal cells was used as an internal, positive control. Typical Rb nuclear staining is shown in Figures 1C and D.

The expression of cyclin D1 was determined by immunohistochemistry in 18 cases of VC. We have previously shown that this immunohistochemical technique only detects cells overexpressing cyclin D 1 and does not detect cyclin D1 in normal proliferative cells4' Cyclin D1 immunostain was seen in 11 cases as a nuclear staining. Positive cases represented moder- ately invasive lesions with some dysplastic areas (Figs. 1E and F). The more aggressive cases and the noninva- sive, hyperplastic lesions as well as the normal oral mucosa (five cases) (Fig. 1H) were negative (Table 1).

DISCUSSION In the last few years several tumor suppressor genes have been identified and cloned. Among them, the p53 and Rb genes are the best characterized. The p53 tumor suppressor gene has been found mutated in a wide variety of human cancers, and mutations in this gene are, furthermore, the most common genetic al- teration found in human n e ~ p l a s i a . ~ ~ - ~ ~ Although the function of the p53 gene has not been completely elu- cidated, p53 appears to be a nuclear transcription fac- tor that plays a role in the control of cell proliferation, apoptosis, and maintenance of the fidelity of DNA du- p l i ~ a t i o n . ~ ~ Mutations in the p53 gene increase the stability of the p53 protein and change the specificity of its transcription factor a~tivity.~' The increased sta- bility of p53 results in accumulation of the protein in the nuclei of tumor cells bearing a mutation in the

Since normal levels of p53 are not detected by immunohistochemical studies on paraffin sections, the presence of positive staining has been accepted as an indicator of possible p53 mutations.

In this study, we show protein accumulation in 15 of 29 VC of the oral mucosa. To our knowledge there is no previous report of the occurrence of p53 in these tumors. The presence of p53 gene alterations was, to some extent, unexpected because p53 alterations have been frequently shown to be related to poor prognosis or advanced stages of disease.52 In this regard, VC is

20 CANCER July 1, 1996 I Volume 78 / Number 1

FIGURE 1. Verrucous carcinoma is shown. (A) p53 expression in the basal and suprabasal layer of a well differentiated verrucous carcinoma. (6) p53 expression in an invasive verrucous carcinoma. (W) Rb protein staining in a verrucous carcinoma. (E&F) cyclin D1 expression. (G) Negative p53 staining in normal human oral mucosa. (H) Negative cyclin 01 staining in normal human oral mucosa. All cases were counterstained with hematoxylin.

P53,

considered a very low-degree malignancy lesion that although locally destructive, rarely metastasizes and almost never jeopardizes the life of the ~ a t i e n t . ~ - ~

However, there seems to be marked differences in the timing of p53 alterations among tumors of differ- ent origins. In some cases, i.e., in colon, thyroid, breast, and prostate carcinomas, mutations of this gene appear to be a late event in the development of the n e o p l a ~ i a . ~ ~ ~ ” ~ In contrast, mutations of the p53 gene have been shown to be present in premalignant lesions of skin, larynx, and e ~ ~ p h a g u s . ~ ~ , ~ ~ . ~ ~ * ~ ~ Our re- sults are also consistent with previous studies carried out in basal cell carcinomas (BCCs), a tumor of the skin, which similarly to VC, may be locally very aggres- sive but not metastatic. Rady et al.55 found heterozy- gous mutations in 50% of BCCs and Shea et al.56 found protein accumulation in 30 of 36 specimens. The re- sults of our study taken with the data from those 2 studies indicate that p53 alteration in squamous epi- thelia is an indicator of local neoplastic growth but does not characterize the evolution to a more malig- nant phenotype in a tumor.

The Rb gene was the first recessive human cancer- susceptibility gene i s ~ l a t e d . ~ ~ - ~ ~ Although originally found to be altered in retinoblastoma, it has now been shown to be involved in the pathogenesis of several other tumors, including prostate carcinomas, osteo- sarcomas, and bladder carcinoma^.^^^^^^^' In this study, the expression of Rb was studied in a selected number of VCs using the same antibody. As shown previously, positive staining of the nucleus using this antibody is an indicator of a functional Rb gene, as tumors pre- senting homozygous mutations of the gene do not nor- mally show expression of the All the cases of VC studied showed a normal staining of the antibody, suggesting that alteration of this tumor suppressor gene does not play a role in the pathogenesis of VC. Similarly, Rb alterations do not seem to be common in more aggressive forms of head and neck SCC. Yo0 et al. have shown that although loci surrounding Rb in chromosome 13 showed high frequency of Loss of Heterozygosity (LOH), Rb alterations are restricted to a small number of cases suggesting that there is another tumor suppressor gene in chromosome 13 which is the actual target of the LOH.62

The involvement of cell cycle regulatory protein, e.g., cyclin D1, CDKs, CDK inhibitors, in cancer has only been recognized in the last few years.32 Derange- ment of the normal expression of cyclin D1 has been shown in a wide variety of turn or^.^^-^^ In head and neck carcinomas, the cyclin D1 gene was shown to be frequently amplified and ove rexpre~sed .~~~”-~~

In this study we have shown that 61% of the VCs express cyclin D1 as determined by immunohisto- chemical methods. Several laboratories have used im-

Rb, Cyclin in Verrucous CarcinomalGimenez-Conti et al. 21

munohistochemical methods to detect overexpression of cyclin D1 in breast cancer, lymphoma, and head and neck carcinoma^.^^*^^-^^ We have previously used immunohistochemical staining to show overex- pression of cyclin D1 protein in mouse skin experi- mental tumors that had shown to overexpress cyclin D1 by Northern blot and immunopre~ipitation.~’~~~

The absence of alterations at the level of Rb pro- tein in VC may be related to overexpression of cyclin D1. It has been postulated that the Rb function may be abrogated as hyperphosphorylation of the Rb pro- tein by overactivation of the cyclin D1-CDK4 complex or loss of the CDK4 regulator p16.7’.72 Alternatively, the Rb function may be lost as a result of HPV infection. The E7 gene of several HPV’s interact with the Rb pro- tein releasing the E2F transcription HPV have been frequently found in VCZm3 and may represent another form of functional Rb inactivation in these tumors.

It is presently unclear why the neoplasms devel- oping from cells with the potential to form squamous epithelia may produce either fully malignant tumors, i.e., SCCs, or tumors with moderate malignant poten- tial that very rarely develop into more malignant le- sions, i.e., VC and BCC. It is possible that alterations of critical genes in early stages of tumor development may be the determinant in the type of tumors. Alterna- tively, it is possible that different target stem cells are involved in the neoplasia of squamous epithelia. Fu- ture studies are necessary to fully understand the na- ture of malignant progression in this neoplasia.

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