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© 2000 American Society for Clinical Oncology p53 Gene Status and Response to Platinum/Paclitaxel-Based Chemotherapy in Advanced Ovarian CarcinomaFrom the Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan; Istituto di Clinica Ostetrica e Ginecologica, Università Cattolica Sacro Cuore, Rome; and Ospedale San Gerardo, Monza, Italy. Address reprint requests to Silvana Pilotti, MD, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy; email pilotti@ istitutotumori.mi.it.
PURPOSE: The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. PATIENTS AND METHODS: Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. RESULTS: Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the patients with mutant p53 tumors, 25 patients (86%) responded to chemotherapy. Only nine (47%) of 19 patients with wild-type p53 tumors responded to the same treatment. The overall response rate and the complete remission rate were significantly higher among patients with mutant p53 tumors than among patients with wild-type p53 tumors (P = .008). Most of the tested tumors not associated with complete remission (10 of 12 tumors) were also characterized by microsatellite instability. The complete remission rate was higher among patients with tumors without microsatellite instability (five of seven patients). CONCLUSION: In contrast to the limited efficacy of treatment with paclitaxel in combination with standard platinum doses against wild-type p53 ovarian tumors, patients with mutant p53 ovarian tumors were more responsive to paclitaxel-based chemotherapy. The pattern of response to chemotherapy containing paclitaxel is different from that associated with high-dose cisplatin therapy. Determining p53 mutational status can be useful in predicting therapeutic response to drugs effective in ovarian carcinoma.
A LARGE NUMBER of cytotoxic agents, including platinum compounds, antimicrotubule agents, alkylating agents, and topoisomerase inhibitors, have been used in the treatment of advanced ovarian carcinoma.1 Combination platinum-paclitaxel chemotherapy has become a standard treatment for advanced-stage disease.2 Outcome is significantly improved with this treatment compared with other platinum-containing combinations. However, the cellular basis of the efficacy of platinum-paclitaxel combinations remains to be defined. In particular, the ability of taxanes to overcome cisplatin resistance could reduce the development of cisplatin-resistant cell populations. An understanding of the biologic or molecular profile of human carcinoma cells, exquisitely responsive to the different classes of agents, could lead to the design of treatment regimens that are more effective in different subgroups of patients. Several lines of evidence support the concept that a major mode of drug-induced cytotoxicity in sensitive cells is the induction of apoptosis.3,4 There is evidence that the wild-type p53 gene product is involved in cellular response to a number of cytotoxic insults, through modulation of cell cycle regulation, DNA repair, and activation of pathways leading to apoptosis.5 Thus, inactivation of the p53 gene could confer resistance to cisplatin and other DNA-damaging agents as a consequence of a reduced cell susceptibility to activate the apoptotic response. Consistent with this hypothesis is the observation that missense mutations are associated with cisplatin resistance in a clinical setting.6 In contrast, in a preliminary study, patients with mutant p53 tumors were found to be responsive to paclitaxel-based therapy.7 Again, the finding is consistent with the peculiar mechanism of action of taxanes (ie, alterations of microtubule function), because the presence of a functional p53 gene is not required for induction of apoptotic cell death by antimicrotubule agents. Cellular pharmacology studies support the concept of an increased sensitivity of mutant p53 cells to taxanes, as a consequence of an accumulation of treated cells in the G2-M phase.8 The efficacy of taxanes against mutant p53 ovarian carcinoma may also have relevant clinical implications, in light of the evidence that p53 mutation is a poor prognostic marker for this tumor.9 To better document the role of p53 status in clinical response in ovarian carcinoma, we further examined the relationships between p53 gene mutations and response to induction chemotherapy containing paclitaxel and platinum compounds in patients with advanced disease. In addition, defects in the DNA mismatch repair system were assessed by analysis of microsatellite instability, because alterations in this system could contribute to resistance.
Patient Characteristics In this retrospective study, we studied 48 previously untreated patients with advanced ovarian cancer. The cases were consecutive series of cases from three independent Italian institutions (20 cases from the Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan; 13 cases from the Ospedale San Gerardo, Monza; and 15 cases from the Università Cattolica Sacro Cuore, Rome). Patients meeting the following criteria were included: treatment with first-line therapy that included paclitaxel and a platinum compound given at a standard dose, adequate sampling of the primary tumor before chemotherapy, and adequate evaluation of tumor stage and response to chemotherapy. Patients were not included in the study if treatment was inadequate, tumor specimens were inadequate for molecular analysis, or the patients were not assessable for tumor response. There were no differences between patients or treatments in the institutions involved in the study.
All patients underwent exploratory laparotomy to determine the feasibility of cytoreduction. Infiltration of the upper gastrointestinal tract and/or the major vessels precluded primary cytoreduction in 12 cases in which only biopsies were performed to obtain a pathologic diagnosis (25%). In 36 cases (75%), it was possible to proceed to surgical removal of tumor masses, along with total abdominal hysterectomy, adnexectomy, radical omentectomy, and appendectomy. In 15 cases, additional surgery (intestinal resection and diaphragm stripping) was required. Radical pelvic and para-aortic lymphadenectomy was performed in all patients undergoing primary cytoreduction who had absent or microscopic residual disease or residual tumors measuring less than 2 cm. Clinicopathologic characteristics of the patient population are summarized in Table 1. Within 2 to 3 weeks of surgery, all patients underwent four to six cycles of induction therapy including a platinum compound (total cisplatin dose
Molecular Analysis of p53 Genomic DNA was extracted from frozen specimens when available (16 cases). In the remaining 32 cases, DNA was extracted from microdissections of 7-µm methylene blue–stained sections obtained from formalin-fixed, paraffin-embedded tissue. In 21 of the latter cases, more than one lesion was available for analysis: 19 cases of bilateral ovarian carcinoma in which tissues were derived from both ovaries and (in nine cases) the metastasis, one case of an ovarian carcinoma in the right ovary and a luteoma in the left ovary synchronous with an endometrioid tumor (patient no.14 [Table 2 ]), and one case (patient no. 4) in which samples from the ovarian carcinoma and the metastasis were available.
All tissue samples were screened by nested polymerase chain reaction (PCR)–single-strand conformational polymorphism (SSCP) for the presence of p53 mutations in the most frequently affected exons (exons 5 through 8) of the gene. The assay was extended to exon 9 in specimens in which no p53 mutation was revealed in exons 5 through 8. DNA extraction and purification were performed using standard procedures (proteinase K digestion followed by spin-column purification). Nested PCR-SSCP and direct DNA sequencing were performed as previously described.10
Immunocytochemistry
Microsatellite Instability Analysis
Statistical Analysis
Response to Therapy Twenty-five (52%) of the 48 patients achieved a complete remission and nine (19%) a partial remission, for a 71% response rate (Table 2). Pathologic complete response was documented in 13 (27%) of the 48 patients. In 14 patients (five with stable disease and nine with progressive disease), disease was considered refractory to therapy for lack of a significant response.
Frequency and Spectrum of p53 Mutations
The position and incidence of the mutations were distributed as follows: Seven mutations were located in exon 5, four in exon 6, 10 in exon 7, seven in exon 8, and one in exon 9. Missense mutations resulting in an amino acid substitution were detected in 27 (93%) of 29 mutations and included 18 transitions (G:C All of the bilateral ovarian carcinoma with mutant p53 (12 of 19 tumors) carried the same p53 mutation in tissues derived from the left and right ovaries and in tissues (when available) from metastases. Tumor no. 4 carried the same mutation in both of the studied lesions (ovarian lesion and metastasis). Tumor no. 14 showed the same mutation in both neoplastic lesions (lesions in the right ovary and the endometrium), but no mutation was noted in the nonneoplastic left ovarian lesion (luteoma). Tumors no. 37 and 45 carried a neutral polymorphism at codon 213 in exon 6, which does not result in a change in the amino acid sequence (CGA-CGG, Arg-Arg), as previously described.14-16 For this reason, the silent mutation in these tumors was not recorded as a mutation in the correlation specified (in the next sections) with immunocytochemistry, response to chemotherapy, or microsatellite analysis. Fifteen of 27 missense mutations occurred at p53 mutational hot-spot codons (codons 175, 216, 220, 248, 273, and 282)17; four of these codons contain CpG nucleotides. Patients no. 6 and 28 carried the same mutation at codon 216 in exon 6.
Immunocytochemistry
Microsatellite Instability
Relationship Between Response to Therapy and Molecular, Clinical, and Pathologic Features To determine whether there was a relationship between response to chemotherapy and p53 gene status or clinical or pathologic features, we divided tumor responses into two categories: response, which included complete and partial responses, and no response (Table 4). The size of residual tumors seemed to be significantly associated with response to therapy (Tables 4 and 5); the response rate was appreciably lower in patients with bulky (> 2 cm) residual disease than in patients with lesions of less than 2 cm (76% v 84%). Reduction of response rate was more evident for extensive unresectable tumors (42%). Patients with grade 1/2 tumors were apparently less responsive than were patients with less differentiated tumors (P = .03). However, the most relevant observation of this study was a statistically significant association between the mutational status of p53 and response to therapy (P = .008). Twenty-five (86%) of 29 patients with mutant p53 responded, whereas nine (47%) of 19 patients with wild-type p53 achieved an objective response. The association between p53 molecular status and response was maintained in multivariate analysis (P = .024) after controlling for grade of differentiation and residual tumor (Table 5).
Despite a significant association between immunocytochemical positivity for p53 protein and gene mutation (Table 6), p53 protein accumulation was apparently a less significant marker of tumor response (24 [72%] of 33 patients with positive tumors were responsive) (Table 4). Indeed, an appreciable number of wild-type p53 tumors were positive (Table 6). Again, the complete remission rate (the number of pathologic complete responses and complete clinical responses) was markedly higher among patients with mutant p53 tumors (20 [69%] of 29) than among patients with wild-type p53 tumors (five [26%] of 19) (P = .013). With a median follow-up of 33 months, a limited number of deaths was recorded: six among the patients with mutant p53 tumors and three among patients with wild-type p53 tumors. As a consequence, the actuarial survival analysis did not show a significant difference between the subgroups. However, patients with mutant p53 had an appreciably increased progression-free survival time (median progression-free survival time, 26 months among patients with mutant p53 tumors and 9 months among patients with wild-type p53 tumors; P = .09). Relevant to this point is the observation that patients with wild-type p53 tumors who did not achieve complete remission with first-line therapy were in most cases still responsive to a second-line therapy that included DNA-damaging agents (ie, alkylating agents or topoisomerase inhibitors). Thus, it is conceivable that second-line therapy influenced survival in this subgroup of patients.
Examination of the microsatellite instability data revealed a lack of association with p53 status in the small series (Table 7). Microsatellite instability reflects defects of the DNA mismatch repair system, which result in a low level of intrinsic resistance to platinum compounds.12 To determine whether there was a relationship between microsatellite instability and therapeutic outcome, we divided responses into three categories: complete response, partial response, and no response. As listed in Table 7, most patients with tumors without evidence of microsatellite instability achieved a complete response (five of seven patients). Microsatellite instability was detected in tumors of most patients who did not achieve a complete response (10 of 12 patients).
Recent evidence supports the concept that specific alterations of tumor cells, involving the expression of certain oncogenes or inactivation of tumor suppressor genes, may have a critical effect on cellular response to cytotoxic injury.3,4,18 Because alterations may involve cell death pathways, it is conceivable that they might play an important role in determining resistance. Mutations of the p53 tumor suppressor gene represent the most common molecular alterations in ovarian carcinoma; such mutations are detected in more than 50% of patients with advanced disease.19 p53 functions have been linked to cell cycle control, DNA repair, stress response, cell senescence, genomic stability, and apoptosis.5,18 Thus, loss of p53 function could play a role in tumor progression. Relevant to this point is the observation that advanced-stage disease is more frequently associated with drug resistance.20 Consistent with the involvement of wild-type p53 function in response to DNA-damaging agents is the observation that mutations of p53 were associated with lack of response to high-dose cisplatin therapy in ovarian carcinoma patients.6 If p53 inactivation is the most relevant factor in determining drug resistance, it is conceivable that mutant p53 tumors retain sensitivity to agents able to trigger a p53-independent apoptosis. Among such agents, the taxanes are now recognized as promising drugs to be used in combination with platinum compounds.21 The present study involved a series of advanced ovarian carcinoma patients with typical clinicopathologic characteristics, comparable with those in other studies.2 In this study, the efficacy of the standard paclitaxel-platinum combination chemotherapy, at least in terms of objective response (71%) and pathologic complete response (27%), corresponded to the clinical results of other studies using such a combination.2 The present investigation provides evidence that paclitaxel-based regimens including standard doses of platinum are effective in the treatment of mutant p53 tumors, thus confirming our preliminary observations obtained in a small series.7 The overall response rate was significantly higher among patients with mutant p53 tumors than among patients with wild-type p53 tumors. This finding is consistent with preclinical evidence of increased sensitivity of ovarian carcinoma cells after inactivation of p53.22,23 The molecular basis for sensitization of ovarian carcinoma cells to antimicrotubule drugs after inactivation of p53 is still unknown. Changes in expression of microtubule-stabilizing proteins or mitotic checkpoint proteins and/or alterations in cell cycle progression as a consequence of loss of p53 function could play a role in enhancing a p53-independent pathway of apoptosis.8,23 Such a pathway still involves the Bcl-2 protein. Inactivation of p53 causes overexpression of Bcl-2, which is under the negative control of p53.24 Because high levels of Bcl-2 may be a resistance mechanism for genotoxic antitumor agents through inhibition of apoptosis,25 the ability of paclitaxel to induce phosphorylation of Bcl-2 could explain the efficacy (and likely the synergistic interaction) of paclitaxel and its combination with platinum compounds in mutant p53 tumors.23 In addition, the pattern of response is consistent with the peculiar p53 mutational profile of ovarian carcinoma, which shows no changes in the various tumor sites of the same patient. The observation suggests that the disease and disease spread both represent a clonal expansion. In the present study, molecular analysis of p53 gene mutations was restricted to the most frequently affected exons.6 Although there may have been a marginal incidence of mutations in exons not studied, the frequency of mutations found is consistent with previous findings in studies of advanced disease.17 An unexpected finding of our study was the relatively low response rate among patients with wild-type p53 tumors. Of the 19 patients with wild-type p53 tumors, only five (26%) achieved a complete remission and four a partial remission, for an overall 47% response rate. This outcome was appreciably poorer than that in our previous study,6,26 in which a high-dose cisplatin treatment (40 mg/m2 for 4 consecutive days, four to five courses) produced a 78% response rate among patients with wild-type p53 tumors (14 of 18 patients, including 11 patients who achieved a complete remission) despite unfavorable clinicopathologic characteristics (ie, bulky residual or extensive disease).26 Given the use of standard doses of platinum compounds in the present study, a tentative explanation for the moderate efficacy of the chemotherapy containing platinum and paclitaxel, in contrast to the marked efficacy of high-dose cisplatin therapy,6,26,27 in patients with wild-type p53 tumors could be that the dose of the DNA-damaging agent was inadequate to trigger an efficient p53-dependent apoptosis. If this hypothesis is correct, full doses of DNA-damaging agents (platinum and alkylating agents) should be preferred in first-line therapy for ovarian carcinoma with wild-type p53. The results also suggest that the efficacy of the combination of taxanes with platinum compounds2 can be ascribed to the exquisite efficacy of the combination against mutant p53 tumors and that the use of paclitaxel in the treatment of wild-type p53 tumors may be questionable. A number of other clinicopathologic and biologic factors may have influenced the pattern of response. Given the well-known heterogeneity of advanced ovarian carcinoma, the pattern of tumor response likely reflects the involvement of a number of established prognostic factors. In particular, a significant association was found in our study between response and degree of differentiation (P = .03) or residual tumor (P = .09). Cases of resistant wild-type p53 tumors included four cases of relatively poorly responsive histologic types (eg, mucinous and clear-cell carcinoma) and one case with pleural effusion. With regard to mucinous tumors, the low response rate can also be ascribed to the higher frequency of Ki-RAS mutations with respect to p53 mutations in this tumor subtype.28 Given these considerations and the limitations of the design of our study, p53 status cannot be regarded as the sole determinant of response. For example, it is likely that other factors are responsible for the level of resistance in patients with tumors carrying wild-type p53, because, as observed in our previous study,6 no precise correlation was found in this subset of patients between complete remission and presence of wild-type p53. On the basis of this observation, we performed a detailed analysis of microsatellite instability in 30 tumors to investigate the functionality of the DNA mismatch repair pathway. The complete remission rate was higher (five of seven cases) among patients with tumors lacking microsatellite instability. In contrast, of 12 patients who did not achieve a complete response, 10 showed microsatellite instability. The observation is consistent with a role of alterations of DNA mismatch repair in the development of platinum resistance. Indeed, resistance to platinum compounds has been ascribed at least in part to loss of DNA mismatch repair as a consequence of failure of the proteins of the repair system to recognize platinum adducts, thus resulting in tolerance of DNA damage.12,29 Because the mechanism related to alterations of the components of the mismatch repair pathway is associated with a low level of resistance, it is likely that an intensification of the platinum dose may provide some therapeutic benefit. Again, the observation is consistent with findings regarding the efficacy of high-dose cisplatin therapy.26,27,30,31 The sensitivity of ovarian carcinoma to DNA-damaging agents cannot be predicted only by p53 functional status, a concept supported by our results; a number of mechanisms involving DNA damage recognition (eg, the mismatch repair system) or DNA repair may be implicated in determining chemosensitivity. The present results may have relevant pharmacologic implications. First, patients with mutant p53 tumors, which are expected to be relatively resistant to platinum compounds,6 seem to be responsive to paclitaxel in combination with platinum compounds. Because the therapeutic success of the combination seems to reflect the efficacy of the agents on different cellular populations carrying different genetic backgrounds rather than the result of a synergistic interaction, p53 status assessment could be useful in selecting subgroups of patients who would benefit from a tailored treatment. If this hypothesis is confirmed, it is conceivable that the optimal therapeutic potential of taxanes and platinum compounds could be achieved with sequential or alternating regimens that allow the use of full doses of the most effective agents. Second, the determination of specific alterations involving recognition and/or repair processes (eg, DNA mismatch repair defects) could allow the selection of patients with tumors in which high-dose platinum could overcome a low degree of resistance. In conclusion, our results provide a rational basis for understanding the heterogeneity of tumor response to effective drugs with a different mechanism of action and for the development of more effective drug strategies based on the molecular profiles of relevant apoptosis-related features.
Supported in part by the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy.
1. Hansen HH, Eisenhauer EA, Hansen M, et al: New cytostatic drugs in ovarian cancer. Ann Oncol 4: S63-S70, 1993
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McGuire WP, Hoskins WJ, Brady MF, et al: Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N Engl J Med 334: 1-6, 1996 3. Zunino F, Perego P, Pilotti S, et al: Role of apoptotic response in cellular resistance to cytotoxic agents. Pharmacol Ther 76: 177-185, 1997[Medline] 4. Hickman JA: Apoptosis and chemotherapy resistance. Eur J Cancer 32A: 921-926, 1996
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Harris CC: Structure and function of the p53 tumor suppressor gene: Clues for rational cancer therapeutic strategies. J Natl Cancer Inst 88: 1442-1455, 1996
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Righetti SC, Della Torre G, Pilotti S, et al: A comparative study of p53 gene mutations, protein accumulation, and response to cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer Res 56: 689-693, 1996 7. Lavarino C, Delia D, Di Palma S, et al: p53 in drug resistance in ovarian cancer. Lancet 349: 1556, 1997 (letter) 8. Wahl AF, Donaldson KL, Fairchild C, et al: Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis. Nat Med 2: 72-79, 1996[Medline] 9. Levesque MA, Katsaros D, Yu H, et al: Mutant p53 protein overexpression is associated with poor outcome in patients with well or moderately differentiated ovarian carcinoma. Cancer 75: 1327-1338, 1995[Medline] 10. Pilotti S, Della Torre G, Lavarino C, et al: Distinct mdm2/p53 expression patterns in liposarcoma subgroups: Implications for different pathogenetic mechanisms. J Pathol 181: 14-24, 1997[Medline] 11. Lavarino C, Corletto V, Mezzelani A, et al: Detection of p53 mutation, loss of heterozygosity and DNA content in fine-needle aspirates of breast carcinoma. Br J Cancer 77: 125-130, 1998[Medline]
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Aebi S, Kurdi-Haidar B, Gordon R, et al: Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Res 56: 3087-3090, 1996 13. Cox DR: Analysis of Binary Data. London, United Kingdom, Methuen, 1970 14. Carbone D, Chiba I, Mitsudomi T: Polymorphism at codon 213 within the p53 gene. Oncogene 6: 1691-1692, 1991[Medline] 15. Elledge RM, Fuqua SAW, Clark GM, et al: Prognostic significance of p53 gene alterations in node-negative breast cancer. Breast Cancer Res Treat 26: 225-235, 1993[Medline] 16. Berns EM, Klijn JG, Smid M, et al: TP53 and MYC gene alterations independently predict poor prognosis in breast cancer patients. Genes Chromosom Cancer 16: 170-179, 1996[Medline]
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Greenblatt MS, Bennett WP, Hollstein M, et al: Mutations in the p53 tumor suppressor gene: Clues to cancer etiology and molecular pathogenesis. Cancer Res 54: 4855-4878, 1994 18. El-Deiry WS: Role of oncogenes in resistance and killing by cancer therapeutic agents. Curr Opin Oncol 9: 79-87, 1997[Medline] 19. McManus DT, Murphy M, Arthur K, et al: p53 mutation, allele loss on chromosome 17p, and DNA content in ovarian carcinoma. J Pathol 179: 177-182, 1996[Medline] 20. Friedlander ML, Dembo AJ: Prognostic factors in ovarian cancer. Semin Oncol 18: 205-212, 1991[Medline]
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Neijt JP: New therapy for ovarian cancer. N Engl J Med 334: 50-51, 1996
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Perego P, Romanelli S, Carenini N, et al: Ovarian cancer cisplatin-resistant cell lines: Multiple changes including collateral sensitivity to Taxol. Ann Oncol 9: 423-430, 1998 23. Zunino F, Cassinelli G, Polizzi D, et al: Molecular mechanisms of resistance to taxanes and therapeutic implications. Drug Resistance Updates 2: 351-357, 1999[Medline] 24. Reed JC, Miyashita T, Takayama S, et al: BCL-2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J Cell Biochem 60: 23-32, 1996[Medline]
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Di Re F, Bohm S, Oriana S, et al: High-dose cisplatin and cyclophosphamide with glutathione in the treatment of advanced ovarian cancer. Ann Oncol 4: 55-61, 1993 27. Bohm S, Oriana S, Spatti G, et al: Dose intensification of platinum compounds with glutathione protection as induction chemotherapy for advanced ovarian carcinoma. Oncology 57: 115-120, 1999[Medline] 28. Caduff RF, Svoboda-Newman SM, Ferguson AW, et al: Comparison of mutations of Ki-RAS and p53 immunoreactivity in borderline and malignant epithelial ovarian tumors. Am J Surg Pathol 23: 323-328, 1999[Medline]
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Fink D, Nebel S, Aebi S, et al: The role of DNA mismatch repair in platinum drug resistance. Cancer Res 56: 4881-4886, 1996 30. Sandercock J, Parmar MKB, Torri V: First-line chemotherapy for advanced ovarian cancer: Paclitaxel, cisplatin and the evidence. Br J Cancer 78: 1471-1478, 1998[Medline] 31. Shapiro JD, Rothenberg ML, Sarosy GA, et al: Dose-intensive combination platinum and cyclophosphamide in the treatment of patients with advanced untreated epithelial ovarian cancer. Cancer 83: 1980-1988, 1998[Medline] Submitted August 13, 1999; accepted June 30, 2000. This article has been cited by other articles:
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
400 mg/m2) and paclitaxel (total paclitaxel dose 
[32P]dCTP (Amersham Pharmacia Biotech, Cologno Monzese, Milan, Italy). The PCR products were separated on a 6% polyacrylamide–7-M urea gel and were visualized using autoradiography. Microsatellite instability was analyzed at four different loci: D2S123, a polymorphic marker localized to chromosome 2p and centromeric to the genomic loci for the mismatch repair genes hMSH2 and hMSH6; D17S250, a marker localized to chromosome 17 in bands 17q11.2 through 17q21 and which maps within the region of the early-onset familial breast and ovarian cancer locus (BRCA1); BAT26, a poly(A) tract in the fifth intron of the hMSH2 gene; and BAT40, a poly(A) tract derived from the intron of the 3-beta-hydroxysteroid dehydrogenase gene. 
A:T in 13 cases and A:T
