Cancer Genet Cytogenet 105:177–181 (1998)
Elsevier Science Inc., 1998
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Correlation between Apoptosis and
Franca Pompetti, Liborio Stuppia, Valentina Gatta,
Giuseppe Calabrese, Gabriella Gamberi, Maria Serena Benassi,
Piero Picci and Giandomenico Palka
Samples of 18 osteosarcomas were examined for the occurrence of apoptosis and
status. Apoptosis was investigated by an in situ nick translation technique on paraffin-embedded sam-
ples. It was found that apoptosis rarely occurs in osteosarcoma at the early stage of disease, whereas it
is frequently activated when tumors are treated with antiblastic drugs. The analysis of the
showed no mutation at diagnosis; whereas, during disease progression, four cases showed
tions. The authors discuss the relation between apoptosis,
status, and therapy. © Elsevier Sci-
ence Inc., 1998
Osteosarcoma (OS) is a bone tumor occurring mainly in
adolescents or young adults . Although preoperative
chemotherapy combined with surgery has improved pa-
tient survival , OS is still a highly malignant tumor with
a poor prognosis.
Molecular studies have found frequent involvement of
tumor suppressor genes in OSs [3–10].
Alterations of several oncogenes also were found in OSs
[10, 11], but their clinical relevance is still unknown.
expression and C
overexpression have been
associated with a poor outcome [10, 12]. None of these
studies correlated genetic mutations with apoptosis, which
controls tumor growth and relates tumor response to ra-
dio- and chemotherapy .
In this study, 18 OS primary tumors and metastases
were investigated for the pattern of apoptosis and the
status, before and after chemotherapy, using in situ
nick translation and p53 PCR-SSCP screening.
MATERIALS AND METHODS
Thirteen males and 5 females with OS, averaging 21 years
(range 5–73) in age, entered this study (patients 1–18). The
diagnosis of OS was based on clinical and histological fea-
tures. According to the grading of Broders et al. , 15 pa-
tients had grade IV OS (patients 1–6, 8–10, 12–16, and 18),
and 3 patients had grade III OS (patients 7, 11, and 17).
Twelve patients (patients 2, 4–6, 10, and 12–18) entered the
protocol of presurgery chemotherapy with methotrexate,
cisplatinum, and adriamycin, according to Bacci et al. .
Another 6 patients did not receive preoperative chemother-
apy (patients 1, 3, 7–9, and 11). After tumor resection, all pa-
tients were treated with postsurgery chemotherapy accord-
ing to Bacci et al. . Response to therapy was evaluated on
the basis of the necrosis present in the primary tumor resec-
tion. Data of investigated patients are reported in Table 1.
All samples consisted of formalin-fixed paraffin-embed-
ded slides. The primary tumor was studied by biopsy in
17 patients (patients 1–11 and 13–18). Tumor resections of
4 patients (patients 12 and 16–18) and metastases of 6 pa-
tients, five from the lung (patients 11–15) and one from
the joint (patient 4), also were examined. In total, 30 sam-
ples were analyzed, as described in Table 1.
In Situ Nick Translation
This method was performed as described by Stuppia et al.
, with modifications. Briefly, slides of formalin-fixed,
paraffin-embedded samples were deparaffinized and fixed
in 3:1 methanol:acetic acid. Slides were then incubated for
30 minutes at room temperature in a solution containing 2
IU of endonuclease-free DNA polymerase I or terminal
deoxynucleotidyl transferase, 10 mM each of dATP, dCTP,
dGTP, and digoxigenin-dUTP in 50 mM Tris-HCl, pH7.8,
5 mM MgCl
, and 10 mM 2-mercaptoethanol. All reagents
were purchased from Boehringer (Mannheim, Germany).
From the Dipartimento di Scienze Biomediche, Sezione di
Genetica Medica, Università G. D’Annunzio (F. P., L. S., V. G.,
G. C., G. P.), Chieti, Italy; and the Laboratorio di Oncologia, Isti-
tuti Ortopedici Rizzoli (G. G., M. S. B., P. P.), Bologna, Italy.
Address reprint requests to: Prof. Giandomenico Palka, Via B.
Buozzi 93, 65100 Pescara, Italy.
Received August 16, 1997; accepted January 21, 1998.