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versión impresa ISSN 1698-4447
Med. oral patol. oral cir. bucal (Ed.impr.) vol.10 no.5 nov./dic. 2005
Oral squamous cell carcinoma. Cytometric parameters of prognostic interest
Carcinoma oral de células escamosas. Parámetros citométricos de interés pronóstico
Ramón Saiz Bustillo (1), Guadalupe Corchero Martín (2), Belén García-Montesinos Perea (3),
Tomás Gonzalez Terán (2), Sergio Sánchez Santolino (2)
(1) Jefe de Servicio del Servicio de Cirugía Oral y Maxilofacial
(2) Médico Residente del Servicio de Cirugía Oral y Maxilofacial
(3) Médico Adjunto del Servicio de Cirugía Oral y Maxilofacial del Hospital Universitario Marqués de Valdecilla (Santander ).
Universidad de Cantabria
Dr. Ramón Saiz Bustillo
Hospital Universitario Marqués de Valdecilla:
Avd.Valdecilla s/n C.P.39008
Tel:942202528 - Fax:942202726
Received: 25-01-2004 Accepted: 19-02-2005
Saiz-Bustillo R, Corchero-Martín G, García-Montesinos-Perea B, Gonzalez-Terán T, Sánchez-Santolino S. Oral squamous cell carcinoma, cytometric parameters of prognostic interest. Med Oral Patol Oral Cir Bucal
Objetivos: el presente estudio se realizó para encontrar posibles factores pronósticos del Carcinoma oral de células escamosas puesto que es una enfermedad frecuente ( 3 4 % de los tumores malignos ) que origina una gran morbilidad y mortalidad y que justifica cualquier intento que trate de aportar algo para conocer mejor esta patología. Diseño del estudio: hemos realizado un estudio sobre 81 carcinomas orales de células escamosas extraídos del archivo del Hospital Universitario Marqués de Valdecilla ( Santander ) , tratados con el mismo procedimiento , de los cuales en 67 de ellos se realizó citometría de flujo.
Palabras clave : Carcinoma oral de células escamosas , citometría de flujo.
Objectives: the present study was made in order to find possible prognostic factors in oral squamous cell carcinoma, given that it is a frequent disease (3-4% of all malignant tumors) and is the cause of a high morbidity and mortality which justifies any attempt to contribute something towards the understanding of this pathology.
Keywords: Oral squamous cell carcinoma, flow cytometry.
Approximately 3% of malignant tumors originate in the oral cavity. The majority of which correspond to squamous cell carcinomas, and a small percentage to malignant tumors of the salivary glands, lymphoreticular diseases, bone tumors, melanomas, sarcomas, malignant odontogenic tumors and metastases from other locations (1).
The prognosis for patients with this pathology depends on the size, infiltration and location of the lesion, presence or absence of metastatic spread, and to a certain degree the differentiation of the tumor (2). Tumor ploidy, measured through flow cytometry, seems to be another prognostic variable. The technique combines knowledge acquired from microspectrophotometry, the production of monoclonal antibodies (3-6), the development of fluorochromes and computer data processing, which are used to establish a series of cellular parameters. It is an objective and automated method for measuring cellular parameters and thus helps in tumor diagnosis, measuring the DNA content and therefore reflecting chromosomal anomalies (7-9), aneuploidy (10) being a highly sensitive and specific parameter. The DNA content also relates to the prognosis (12-18), in such a way that a DNA index (DNA content of G1 phase tumor cells/DNA content of G1 phase euploid cells) other than 1 is accompanied by a worse prognosis. Likewise, this technique can be applied to the treatment of patients, since in chemotherapy and radiotherapy the response to treatment can be related to the alterations produced in the cell cycle (19).
MATERIAL AND METHODS
For the study, 81 diagnosed cases of OSCC, obtained from the Departamento de Cirugía Maxilofacial del Hospital Universitario Marqués de Valdecilla (Santander), treated curatively with surgery and radiotherapy, and excluding cancer of the lip, were used. Flow cytometry was carried out in 67 cases, the remaining 14 were excluded for having insufficient histological tumor material available, and/or for presenting a variation coefficient higher than 10.
In all cases the resected tumor sample was available, from which representative samples were taken, fixed in formol and embedded in paraffin. All the lymph nodes isolated from the surgical specimen were processed. Sections stained with hematoxylin-eosin were used to typify the tumors. From the macro and microscopic data the TNM classification was determined, establishing the pathological stage. For the flow cytometry, the material was stained in accordance with the method described by Vindelov and Christensen (11). The FACScan flow cytometer (Becton Dickinson), equipped with an argon laser (488-518 nm) was used, with the Cellfit Cellcycle Analysis software version 2.0.2.
The cytometric variables analyzed were: the DNA index, ploidy, S-phase fraction, the proliferative index, and the GO/G1 peak variation coefficient.
The statistical analysis of the data was made using the SPSS statistical package (release 5. 0. 2, 1993):
Descriptive statistics: mean, standard deviation and maximum and minimum values for the quantitative variables, and frequencies and percentages for the qualitative.
Inferential statistics: using the Students t test, analysis of variance (ANOVA), Spearman rank correlation coefficient, proportional contrast test, contingency tables according to the chi-square distribution and the Fisher Exact test.
Survival analysis: following the nonparametric Kaplan-Meier model and log rank test.
Multivariate analysis: cox multiple regression analysis.
Forty-nine of the 67 cases analyzed (73.13%) were diploid, and 18 (26.86%) aneuploid. Of the diploid, 3 were peridiploid; and of the aneuploid, 16 were hyperdiploid and 2 tetraploid. No significant differences were found in the distribution of patients with respect to age, sex, presence or absence of lymph node involvement, or with respect to the average value for the mitotic index in diploid or aneuploid tumors, or with respect to the tumor size or T category.
Cellular proliferative index:
The mean cellular proliferative index for the total of cases was 14.99 ± 8.87, with maximum and minimum values of 4.3 and 39.2 respectively. There were no significant differences between the cellular proliferative index and age, sex, location of the lesion, or T or N categories, although this index was grouped into two categories, less than 25% and more than 25%.
Where the mitotic index was less than 10, the cellular proliferative index was 12.638 (7.216, d.t.); while for tumors with a mitotic index equal to or greater than 10 it was 17.744 (10.140, d.t.), with p<0.05 and T=2.1211.
With respect to the clonal category, the majority of diploid tumors presented a cellular proliferative index of less than 25% (95.65% of diploid tumors), in contrast to the aneuploid, in which a proliferative index greater than 25% was more frequent, (p<0.001 and chi-square=19.2212).
The mean S-phase fraction (SPF) in the tumors studied was 11.67% ± 8.15%, with maximum and minimum values of 3.4% and 38.4% respectively. Grouping the percentage of cells in S-phase into classes it was observed that most cases corresponded to an SPF between 5 and 10%.
No differences in the SPF were found in relation to the age or sex of the patient, tumor location, or T or N categories.
The mean mitotic index in those tumors with an SPF of less than 20 was 10.708 (9.293 d.t.), while in tumors with an SPF equal to or greater than 20 it was 16.860 (13.035 d.t.), these differences were not significant.
Significant differences were found in the clonal category between the S-phase of diploid and aneuploid tumors, (p<0.001 and chi-square = 24.16), in such a way that the diploids presented an SPF of less than 20 in 95.65% of cases, while in the aneuploids the SPF was greater than 20 in 72.72% of cases. The mean SPF in the diploid tumors was 8.77% ± 4.86, while in the aneuploids this was 23.8% ± 8.11, (p<0.001 and T = 5.8939). Likewise, those tumors with an SPF of less than 20 exclusively present a cellular proliferative index of less than 25, while the tumors with a cellular proliferative index greater than 25% present an elevated SPF in 100% of cases, (p<0.001 and chi-square = 43.6905). The average percentage of S-phase cells in those tumors with a cellular proliferative index of less than 25% was 8.78% ± 4.43, while in those cases with a proliferative index of greater than 25% this percentage of S-phase cells was 27.1% ± 5.67, (p<0.001 and T=10.8704).
None of the cytometric variables studied presented any relationship with the appearance of local relapse, the later appearance of distant metastasis, or with survival.
The study of prognostic factors in oncologic processes is of great interest because it allows us to know the natural history of the disease, makes it possible to form homogenous groups of patients in clinical trials, and facilitates the design of selective clinical trials. Furthermore, it explores possible interactions between the prognostic variables and the treatment, and allows the prediction of prognosis in patients and can help to explain the variations detected in the survival between groups. In this way, it influences the therapeutic strategy, improves the stratification of patients in random studies and makes the comparison of results possible.
From the results obtained in our study, we cannot confirm that flow cytometry is a technique with any reliable prognostic value, although some data have certain significance. On relating survival with the parameters obtained by flow cytometry, we have not found any that provide significant data.
The results obtained by flow cytometry serve as a guide, but are insufficient for us to confirm that this technique can be used to determine, with certainty, the aggressive capacity of an epidermoid carcinoma in the oral cavity.
Depending on the location and extension of the primary tumor and the condition of the lymph nodes, treatment of cancer of the oral cavity maybe only surgical, exclusively through radiotherapy, or a combination of both (20, 21).
The wide discrepancy with regard to the type of treatment for tumors of the oral cavity is expressed by the large number of existing therapeutic protocols, which necessitates the continued search for predictive variables of its behavior.
In supraglottic laryngeal carcinoma (22) the lymph node involvement is associated to variables such as the grade of differentiation (Broders), with a higher incidence of lymph node involvement in undifferentiated tumors. Other variables such as the broad pattern or the presence of a peri-tumoral lymphoplasmacytic infiltrate are related with the absence of lymph node metastasis. The presence of tumor necrosis, on the other hand, is related to the appearance of affected lymph nodes. The stromal reaction and the cytologic grade would not, for these authors, be useful parameters in identifying the appearance of lymph node involvement.
Coinciding with these authors, the existence of lymph node involvement, in our case, has been found in tumors with significant local extension, poorly differentiated, with absent or light inflammatory tumor infiltrate, or the presence of tumor necrosis.
The prognostic value with regard to survival and the disease-free interval of the stromal inflammatory reaction and the vascular space invasion has been demonstrated in tumors of the cervix (23). In this study, the stromal reaction presented as an independent prognostic variable. An intense stromal reaction is related to a lesser lymph node involvement and to a lower recurrence, being more intense in microinvasive tumors. The grade of stromal reaction, with respect to the type of infiltrate, its quantity and necrosis, has not been related to survival in tumors of the head and neck in the orofacial area (24). Likewise, in tumors such as that of the lip, the cellular composition and the intensity of the stromal reaction have not presented any prognostic value either.
In recent years efforts have increased to relate the analysis of the DNA content and the prognosis in the various solid tumors.
The essence of DNA analysis is the measuring of the DNA content in a large number of individual cells and the construction of a histogram representing the distribution, within the study, of the cell population.
At the molecular level, various factors determine the behavior of a tumor. The identification of quantifiable variables at this level, which directly or indirectly condition the tumor biology, could help the clinician to determine the state of risk of a patient and thereby select the best therapeutic options.
Aneuploidy implies the presence of an abnormal quantity of genetic material and carries a more unfavorable prognosis as the DNA index increases.
Cytometric studies carried out on tumors of the penis indicate that a high DNA index can indicate an increased risk of progression of the disease, so these patients would therefore be candidates for a more aggressive treatment (25).
The majority of tumors have aneuploid cellular populations and those that are only slightly hyperdiploid have a better prognosis.
Authors such as Janot et al. (26) have not found any significance in flow cytometry with regard to prognosis in tumors of the head and neck.
In tumors such as those of the major salivary glands, aneuploidy is related to ill-defined margins by nuclear magnetic resonance, and with a worse prognosis (27).
Although the relationship between ploidy, the recurrence of disease and survival has not been definitively demonstrated, in flow cytometry, an elevated S-phase fraction indicates an increased probability of recurrence in diploid tumors, principally in the early stages of the disease.
In early-stage tumors of the buccal mucosa, (T1 and T2 tumors), aneuploidy is the major predictor for the biological aggressive behavior of the tumor; its presence would indicate the necessity for a more aggressive treatment. In these tumors of the buccal mucosa the presence of aneuploidy indicates a recurrence of 70%, while in the diploids it is approximately 5%.
It has been observed in cervical neoplasias previously treated with radiotherapy that the aneuploid tumors are more sensitive to treatment (28).
The relationship between the S-phase fraction and prognosis has been observed in sarcomas, in such a way that an increased SPF is associated with a worse prognosis (29), although not in tumors such as that of cancer of the esophagus.
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