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Asia-Pacific Journal of Clinical Oncology 2014
doi: 10.1111/ajco.12310
ORIGINAL ARTICLE
BRCA1 mutation site may be linked with nuclear DNA ploidy in BRCA1-mutated ovarian carcinomas Morteza AGHMESHEH,1,2 Akshat SAXENA1 and Farshid NIKNAM1 1 Illawarra Cancer Care Centre, Wollongong Hospital, Wollongong, New South Wales, Australia, 2Peter MacCallum Cancer Centre, Research Division, The Kathleen Cuningham Consortium for Research into Familial Breast Cancer, East Melbourne, Victoria, Australia
Abstract Aims: BRCA1 has a role in maintaining normal nuclear DNA content during cell division and its inactivation may result in DNA aneuploidy and cancer progression. BRCA1-linked breast cancers are more aneuploid and have a worse prognosis, but this has not been elucidated in ovarian cancers. This study explores the potential difference in ploidy status between BRCA1-mutated and sporadic ovarian carcinomas. It also explores the potential association between BRCA1 mutation site and DNA ploidy status. Methods: This study compared DNA ploidy status of tumor blocks from 23 BRCA1-mutated ovarian carcinomas with that of 23 sporadic ovarian carcinomas matched for histologic subtype, patient age, stage and grade. DNA content of the nuclei was measured by Feulgen–Schiff staining followed by image cytometry and compared. Results: BRCA1-linked tumors with a stop codon closer to the N-terminal (between 1 and 500 aa; 6/6, 100%) had a significantly higher frequency of nondiploidy compared with those with stop codon above 500 aa (7/12, 58%) (P = 0.033). A diploid peak was detected in 28% of BRCA1-mutated ovarian cancers and in 33% of sporadic ovarian cancers. Conclusions: The present study concluded that ovarian tumors with mutations closer to the N-terminal of BRCA1 may have a higher risk of DNA aneuploidy. There is no significant difference between BRCA1mutated and sporadic ovarian carcinomas with respect to the DNA content. Key words: aneuploidy, BRCA1, DNA content, image cytometry, ovarian cancer, ploidy.
INTRODUCTION Previous studies have suggested a link between centrosome duplication, centrosome amplification defects, aggressive tumors and aneuploidy. BRCA1 is a tumor suppressor gene with a critical role in double-stranded DNA damage repair, which results in genomic stability. BRCA1 and p53, apart from their main function as tumor suppressor genes, play a significant role in con-
Correspondence: Prof Morteza Aghmesheh, MBBS, PhD, Department of Medical Oncology, The Wollongong Hospital, Crown Street, Wollongong, NSW 2500, Australia. Email: [email protected] Conflict of interest: The authors declare that there are no conflicts of interest Accepted for publication 7 September 2014.
© 2014 Wiley Publishing Asia Pty Ltd
trolling centrosome duplication by controlling the mitotic spindle assembly.1–4 Inhibition of BRCA1 function in mammary epithelial cells is acutely followed by DNA aneuploidy in these cells.5 BRCA1 localizes to centrosome along with other proteins that regulate the G2-M check point, including p53 and retinoblastoma (RB).6,7 Xu et al. showed in vitro that deletion of exon 11 of BRCA1 in fibroblast cells leads to unequal chromosome segregation, abnormal nuclear division and aneuploidy.1 Similarly, BRCA1-deficient cells obtained from knockout mouse embryos develop extra centrosomes and aneuploidy,1,8 suggesting that BRCA1 may play a role in maintaining normal DNA content during cell division, and as a corollary to this, that mutations in BRCA1 may lead to aneuploidy. Mutations in BRCA1 are associated with increased incidence of breast, fallopian and ovarian cancer.9–12 BRCA1 mutation-induced
2
genomic instability may facilitate the accumulation of multiple gene mutations.13 This could lead to activation of multiple signal pathways that play key roles in carcinogenesis, metastasis, prognosis and drug resistance.14–19 Consistently, BRCA1-linked breast cancers are more aneuploid compared with sporadic breast cancers20,21 and have a worse prognosis.22 Ploidy has been reported to be of prognostic significance in ovarian cancer and patients with diploid tumors have a significantly better survival than those with aneuploid tumors.23,24 There have been only few reports and only on small number of patients on either studies of ploidy in ovarian cancers from women with germline mutations in BRCA1 or comparing the DNA content of ovarian cancers in such patients with that of sporadic ovarian cancers. The aim of this study was to compare the DNA content in ovarian cancers from women with germline mutations in BRCA1 with sporadic controls to ascertain whether there were differences, and if so, whether there was any correlation with biological behavior.
METHODS Sample collection Paraffin-embedded ovarian tumors from 23 confirmed BRCA1-mutated ovarian cancer patients were collected from different hospitals in Sydney and KConFab. This group of patients was matched for histologic subtype, patient age, stage and grade with 23 ovarian cancer specimens from patients with no significant familial history of breast or ovarian carcinoma, termed sporadic cases.
Measurement of DNA content The tissue sections with more than 80% tumor tissue were used for the preparation of nuclei suspension to avoid contamination with normal tissue. Monolayers were prepared from 1 to 3 of 50-μm section/s using an established method.25 The DNA content of the nuclei was stained using Feulgen–Schiff staining procedure after monolayer preparation of paraffin sections. A Fairfield DNA ploidy system version 1.4.1 (Fairfield Imaging Ltd, Nottingham, UK) was used to process the images, which were stored with 1024 gray levels. The integrated optical density of each nucleus was calculated on the basis of measurements of optical density and area. Background optical density was measured and corrected for each nucleus.
© 2014 Wiley Publishing Asia Pty Ltd
M Aghmesheh et al.
At least 1500 nuclei were collected into galleries: lymphocytes, plasma cells and fibroblast nuclei were selected as reference cells. Cut, overlapped and pyknotic nuclei were excluded from the study. DNA histograms for each case were generated by image analysis. The resulting DNA histograms were classified based on their ploidy status (see the following section).
Classification of DNA ploidy The tumor was defined as diploid if only one G0/G1 peak (2c) was present in the histogram, the number of nuclei in the G2 peak (4c) did not exceed 10% of the total number of nuclei and the number of nuclei with a DNA content > 5c did not exceed 1%. A tumor was defined as tetraploid when a G0/G1 peak in the diploid position (4c) was present along with a G2 peak in the octa-position (8c, 8 copies of DNA) or the fraction of nuclei in the tetraploid region exceeded 10% of the total number of nuclei. The presence of a peak in the 8c position together with a G2 peak in the 16c position was an indication of polyploidy for a tumor.25 The tumor was defined as aneuploid when noneuploid peaks were present or the number of nuclei with DNA content > 5c, not representing euploid peaks, exceeded 1%. The histograms related to the DNA content were classified blindly (to the mutation status and clinical data) by Dr. Havard Danielsen. The coefficient of variation (CV) was calculated from the major peak in each case and the CV of the DNA diploid stem line in paraffin-embedded tissues was considered if it was 0.05).
Association of DNA content with stage, grade, histological subtype and patient’s age The majority of cases examined in this study were serous cystadenocarcinoma (93%, 39/42) and there were 2 Table 1 Distribution of DNA ploidy status in 18 BRCA1mutated and 23 sporadic ovarian carcinomas measured by DNA image cytometry
Diploid Tetraploid Aneuploid
BRCA1-mutated cases (%)
Sporadic cases (%)
5/18a (28) 3/18c (17) 10/18e (56)
7/23b (30) 3/23d (13) 13/23f (57)
Fisher’s test, P = 0.565 between a and b; P = 0.779 between c and d; and P = 0.599 between e and f.
Asia-Pac J Clin Oncol 2014
cases of mixed Mullerian tumor and two mixed tumors (serous + endometrial and serous + clear cell carcinomas). A summary of the association of aneuploidy DNA with age, stage and grade in BRCA1-linked and sporadic ovarian carcinomas is provided in Table 2. Of the entire study groups, observed DNA aneuploidy was numerically more common in subjects who were ≥50 years of age (67%, 12/18) compared with those below the age of 50 (48%, 11/23). However, this was not statistically significant (Table 2). There was more DNA aneuploidy in stage III (63%) and stage IV (60%) compared with stage II (17%) ovarian tumors. The difference between stage III and stage II reached statistical significance (19/30 vs 1/6, Fisher’s test, P = 0.043). No significant difference in DNA ploidy was observed among tumors of different histological grades (Table 2).
Association between BRCA1 and nuclear DNA content The association between BRCA1 mutation site and DNA content was investigated in 18 BRCA1-mutated ovarian cancers and their matched sporadic counterparts (Table 3). For statistical analysis, BRCA1-linked cases were divided into four groups according to their mutation site: BRCA1 mutations with stop codon less than 500 (amino acids), those with stop codon between 501 and 1000, stop codons between 1001 and 1500, and finally stop codons above 1500 (Fig. 1). BRCA1 mutation site was marginally correlated with DNA ploidy with more
Table 2 Association of aneuploid DNA with stage and grade in BRCA1-linked and sporadic ovarian carcinomas Number of aneuploid cases
Age (years) A 1294–1333 del 40 1471 C > A 1876 del C 3458 T > G 3450–3453 del CAAG 3450–3453 del CAAG 3875–3878 del GTCT 3875–3878 del GTCT 4184–4187 del TCAA 4184–4187 del TCAA 4184–4187 del TCAA Exon13 duplication Exon13 duplication 5382–5383 ins C
A A A T A A D D A D T A T A D A A D
BRCA1 mutations are ranked from N-terminal (top of table) toward the C-terminal (bottom). A, aneuploid; D, diploid; T, tetraploid.
Figure 1 Potential association between BRCA1 mutation site and DNA ploidy. Schematic view of BRCA1 protein with its N-terminal and C-terminal domains is demonstrated. DNA ploidy status of BRCA1-mutated ovarian cancers with stop codons ranging between 39 and 1829 is illustrated in this schematic view by circle (aneuploid), square (diploid) and triangle (tetraploid). Amino acids 504–803 of BRCA1 protein have been suggested to be a binding site for γ-tubulin with a potential role in DNA ploidy.
aneuploidy in cases having a mutation close to the N-terminal (with small amino acid number), while there were more euploid cases in those with a mutation close to the C-terminal (Kruskal–Wallis test, P = 0.050). Furthermore, a significant difference was noticed between two groups, aneuploid and non-aneuploid (diploid plus tetraploid), when analyzed using the Mann–Whitney U-test (P = 0.029). As indicated in Table 3, there are more aneuploid cases in patients with a stop codon close to the N-terminal of BRCA1 compared with those close to the C-terminal. Amino acids 504–803 of BRCA1 protein
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have been suggested to be a binding site with a potential role in DNA ploidy.27 Since the postulated binding site was between 504 and 803, for statistical purposes, BRCA1-mutated ovarian cancers were divided into two categories: cases with stop codons between 1 and 500 (close to the N-terminal of BRCA1 and prior to potential binding site) versus those with stop codons between amino acids 501 and 1863 (including the binding site) (Fig. 1). It was found that the BRCA1-linked tumors with a stop codon between 1 and 500 aa (6/6, 100%) had a significantly higher frequency of nondiploidy than those
Asia-Pac J Clin Oncol 2014
BRCA1 mutation and DNA content in ovarian cancer
with stop codon above 500 aa (7/12, 58%) (mean ranks: 14.42 vs 8.82; Mann–Whitney U-test, P = 0.033). As Table 3 demonstrates, tumor specimens with identical mutations demonstrated differences in DNA ploidy status.
DISCUSSION We are the first to report that there is significant correlation between BRCA1 mutation site and DNA aneuploidy in ovarian cancer samples. Of note, a specific domain of the BRCA1 protein, amino acids 504–803, was previously reported to be a binding site for γ-tubulin, an essential component of the centrosome.27 This encouraged us to divide BRCA1 mutations into two categories: (i) ovarian cancers with BRCA1 mutations resulting in a stop codon prior to 500 aa: such cases have lost the BRCA1 interaction site for γ-tubulin (amino acids 504–803) and (ii) those with stop codons above 500 aa, which may have lost the function of the interaction site for γ-tubulin. All BRCA1-linked ovarian cancers with a stop codon prior to 500 aa were nondiploid, whereas those with a stop codon above 500 were either diploid or tetraploid/aneuploid. This difference was statistically significant and is consistent with the data described by Hsu and White, suggesting that BRCA1 may be one of the key proteins in maintaining normal DNA content and that amino acids 504–803 provide a potential interaction site for this function of BRCA1.7 Further studies may focus on whether BRCA1related ovarian cancers with stop codons prior to 500 aa have poorer prognosis due to an increased chance of developing aneuploid tumors. What is the key factor involved in DNA aneuploidy of sporadic ovarian cancers if BRCA1 mutation has a role in DNA ploidy of BRCA1-related ovarian cancer? Somatic mutations in the p53 gene are another mechanism suspected of being involved in DNA ploidy status. We did not, however, find any significant association between p53 and DNA ploidy in either BRCA1-related or sporadic ovarian carcinomas in the present study. This is consistent with a previous study by Valverde et al. who found no association between DNA ploidy and p53.28 The other possible mechanism for DNA aneuploidy in sporadic ovarian cancers is BRCA1 inactivation through mechanisms other than mutation such as methylation.29 This is in support of the hypothesis that BRCA1-mutated ovarian cancers and at least a subpopulation of sporadic ovarian cancers develop through similar carcinogenic pathway.
Asia-Pac J Clin Oncol 2014
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No published study has compared the DNA content in matched BRCA1-mutated and sporadic ovarian carcinomas. Our results indicated that there was no significant difference in the DNA content between the small size cohorts of BRCA1-mutated and sporadic ovarian carcinomas. This differs from the findings in BRCA1mutated breast cancers, which have a higher frequency of DNA aneuploidy and poorer prognosis compared with sporadic tumors.20–22 The results of the present study are in agreement with that of Auranen et al., who compared 58 familial ovarian cancer cases (not characterized by genetic testing in the patients) with 532 sporadic ovarian tumors for DNA ploidy by flow cytometry.30 These authors reported 46% aneuploid tumors in the familial group with no significant difference between familial and sporadic cancers. The advantages of the present study compared with Auranen et al.’s study are that ovarian tumor tissues with known BRCA1 mutation status were used in the present study and BRCA1-mutated tumors with their sporadic counterparts were matched for factors with potential influence on DNA ploidy status, namely histologic subtype, patient age, stage and grade.26 In addition, image cytometry used in this study is a more sensitive method of detecting aneuploidy than the flow cytometry used in Auranen et al.’s study.31,32 The lack of difference in DNA ploidy between BRCA1-linked and sporadic ovarian carcinomas may indicate that their carcinogenic pathways are similar. However, the potential “contamination” of the sporadic group in the present study may be a contributing factor to the absence of difference in DNA content between the two groups. BRCA1 mutations in the sporadic cohort of the present study was not tested due to ethical and practical reasons; data from other studies suggest that at least 10% of the sporadic cancers in the present study may have BRCA1 germline mutations.33 The possibility of a type II error (false-negative results due to a small sample size) exists and a study of larger cohorts is required to obtain more definite conclusion since the sample size in the present study is limited. DNA aneuploidy was more common in stage III compared with stage II ovarian cancers and that was statistically significant. There was no detectable difference between stage III and stage IV, possibly because of the limited number of stage IV ovarian cancers in the present study, but it may also indicate that aneuploidy is more important in the progression from stage II to stage III and less important from stage III to stage IV. This result suggests that DNA aneuploidy is more common in advanced ovarian cancers compared with early stages,
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which is in agreement with findings from results from a previous study.26 There was no significant correlation between grade and DNA aneuploidy or between age and aneuploidy in this study. In contrast, Trope and colleagues showed that aneuploid ovarian tumors are more often stage Ic (rather than Ia), poorly differentiated, of serous type, and in patients older than 50 years.26 As the majority of cases in the present study were serous, stage 3, grade 3 and in a younger age group in comparison with previously reported studies of sporadic ovarian cancers, the possibility of detecting any potential correlation between age, stage, histologic subtype or grade, and DNA ploidy was reduced. This study was limited by the sample size. Study of a larger cohort is required to confirm this result. The survival data were not accessible for most of the BRCA1- mutated group, limiting the ability to investigate any association between DNA aneuploidy and prognosis/response to chemotherapy, as well as links between the BRCA1 mutation site and prognosis/ response to chemotherapy. The sporadic group in the present study could not be tested for BRCA1 mutations because of ethical concerns. In conclusion, the present study is the first to demonstrate that ovarian tumors with mutations closer to the N-terminal of BRCA1 have a higher risk of DNA aneuploidy. It is tempting to postulate that BRCA1mutated ovarian carcinomas whose BRCA1 mutations result in a stop codon prior to 500 aa may behave differently in their prognosis; however, this needs to be formally tested in a larger prospectively collected sample. This study also found for the first time that there is no significant difference between BRCA1mutated and sporadic ovarian carcinomas with respect to the DNA content. This is in contrast to ploidy data in breast cancer where BRCA1-linked tumors were shown to be more aneuploid compared with their sporadic counterparts.
ACKNOWLEDGMENTS This study was supported by substantial donations from Mr. Steve Eckowitz and the Farmoz Company and in part by the National Institutes of Health (USA) (NIH CA 18119). KConFab has been funded by the Kathleen Cuningham Foundation, National Breast Cancer Foundation, National Health and Medical Research Council, Anti-Cancer Council of Victoria, Anti-Cancer Foundation of South Australia, Cancer Foundation of Western
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M Aghmesheh et al.
Australia and Queensland Cancer Fund and NSW Cancer Council.
REFERENCES 1 Xu X, Weaver Z, Linke SP et al. Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol Cell 1999; 3: 389–95. 2 Deng C, Brodie SG. Roles of BRCA1 and its interacting proteins. Bioessays 2000; 22: 728–37. 3 Kramer A, Neben K, Ho AD. Centrosome replication, genomic instability and cancer. Leukemia 2002; 16 (5): 767–75. 4 Tarapore P, Horn HF, Tokuyama Y, Fukasawa K. Direct regulation of the centrosome duplication cycle by the p53p21Waf1/Cip1 pathway. Oncogene 2001; 20 (25): 3173– 84. 5 Schlegel BP, Starita LM, Parvin JD. Overexpression of a protein fragment of RNA helicase A causes inhibition of endogenous BRCA1 function and defects in ploidy and cytokinesis in mammary epithelial cells. Oncogene 2003; 22 (7): 983–91. 6 Deng CX. Tumorigenesis as a consequence of genetic instability in BRCA1 mutant mice. Mutat Res 2001; 477 (1,2): 183–9. 7 Hsu LC, White RL. BRCA1 is associated with the centrosome during mitosis. Proc Natl Acad Sci U S A 1998; 95 (22): 12983–8. 8 Shen SX, Weaver Z, Xu X et al. A targeted disruption of the murine BRCA1 gene causes gamma-irradiation hypersensitivity and genetic instability. Oncogene 1998; 17 (24): 3115–24. 9 Vicus D, Rosen B, Lubinski J et al. Tamoxifen and the risk of ovarian cancer in BRCA1 mutation carriers. Gynecol Oncol 2009; 115 (1): 135–7. 10 Vicus D, Finch A, Cass I et al. Prevalence of BRCA1 and BRCA2 germ line mutations among women with carcinoma of the fallopian tube. Gynecol Oncol 2010; 118 (3): 299–302. 11 South SA, Vance H, Farrell C et al. Consideration of hereditary nonpolyposis colorectal cancer in BRCA mutation-negative familial ovarian cancers. Cancer 2009; 115 (2): 324–33. 12 Nkondjock A, Ghadirian P. Epidemiology of breast cancer among BRCA mutation carriers: an overview. Cancer Lett 2004; 205 (1): 1–8. 13 Venkitaraman AR. Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment. Annu Rev Pathol 2009; 4: 461–87. 14 Gislette T, Chen J. The possible role of IL-17 in obesityassociated cancer. ScientificWorldJournal 2010; 10: 2265– 71. 15 Mehta K, Osipo C. Trastuzumab resistance: role for Notch signaling. ScientificWorldJournal 2009; 9: 1438–48.
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16 Chen J, McMillan NAJ. Molecular basis of pathogenesis, prognosis and therapy in chronic lymphocytic leukaemia. Cancer Biol Ther 2008; 7 (2): 174–9. 17 Chen J. The Src/PI3K/Akt signal pathway may play a key role in decreased drug efficacy in obesity-associated cancer. J Cell Biochem 2010; 110 (2): 279–80. 18 Hwang JJ, Ghobrial IM, Anderson KC. New frontiers in the treatment of multiple myeloma. ScientificWorldJournal 2006; 6: 1475–503. 19 Huveneers S, Danen EH. The interaction of SRC kinase with beta3 integrin tails: a potential therapeutic target in thrombosis and cancer. ScientificWorldJournal 2010; 10: 1100–5. 20 Marcus JN, Watson P, Page DL et al. Hereditary breast cancer: pathobiology, prognosis, and BRCA1 and BRCA2 gene linkage. Cancer 1996; 77 (4): 697–709. 21 Johannsson OT, Idvall I, Anderson C et al. Tumour biological features of BRCA1-induced breast and ovarian cancer. Eur J Cancer 1997; 33 (3): 362–71. 22 Stoppa-Lyonnet D, Ansquer Y, Dreyfus H et al. Familial invasive breast cancers: worse outcome related to BRCA1 mutations. J Clin Oncol 2000; 18 (24): 4053–9. 23 Gajewski WH, Fuller AF Jr, Pastel-Ley C, Flotte TJ, Bell DA. Prognostic significance of DNA content in epithelial ovarian cancer. Gynecol Oncol 1994; 53 (1): 5–12. 24 Trope C, Kaern J. DNA ploidy in epithelial ovarian cancer: a new independent prognostic factor? Gynecol Oncol 1994; 53 (1): 1–4. 25 Pradhan M, Abeler VM, Danielsen HE, Trope CG, Risberg BA. Image cytometry DNA ploidy correlates with
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histological subtypes in endometrial carcinomas. Mod Pathol 2006; 19 (9): 1227–35. Trope C, Kaern J. DNA ploidy in epithelial ovarian cancer: a new independent prognostic factor. Gynecol Oncol 1994; 53: 1–4. Hsu L-C, Doan TP, White RL. Identification of a gammatubulin-binding domain in BRCA1. Cancer Res 2001; 61 (21): 7713–18. Valverde JJ, Martin M, Garcia-Asenjo JA, Casado A, Vidart JA, Diaz-Rubio E. Prognostic value of DNA quantification in early epithelial ovarian carcinoma. Obstet Gynecol 2001; 97 (3): 409–16. Senturk E, Cohen S, Dottino PR, Martignetti JA. A critical re-appraisal of BRCA1 methylation studies in ovarian cancer. Gynecol Oncol 2010; 119 (2): 376–83. Auranen A, Grenman S, Klemi PJ. Immunohistochemically detected p53 and HER-2/neu expression and nuclear DNA content in familial epithelial ovarian carcinomas. Cancer 1997; 79 (11): 2147–53. Kaern J, Wetteland J, Trope CG et al. Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer. Cytometry 1992; 13 (3): 314–21. Reles AE, Gee C, Schellschmidt I et al. Prognostic significance of DNA content and S-phase fraction in epithelial ovarian carcinomas analyzed by image cytometry. Gynecol Oncol 1998; 71 (1): 3–13. Risch HA, McLaughlin JR, Cole DEC et al. Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer. Am J Hum Genet 2001; 68: 700–10.
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Asia-Pacific Journal of Clinical Oncology 2014
doi: 10.1111/ajco.12310
ORIGINAL ARTICLE
BRCA1 mutation site may be linked with nuclear DNA ploidy in BRCA1-mutated ovarian carcinomas Morteza AGHMESHEH,1,2 Akshat SAXENA1 and Farshid NIKNAM1 1 Illawarra Cancer Care Centre, Wollongong Hospital, Wollongong, New South Wales, Australia, 2Peter MacCallum Cancer Centre, Research Division, The Kathleen Cuningham Consortium for Research into Familial Breast Cancer, East Melbourne, Victoria, Australia
Abstract Aims: BRCA1 has a role in maintaining normal nuclear DNA content during cell division and its inactivation may result in DNA aneuploidy and cancer progression. BRCA1-linked breast cancers are more aneuploid and have a worse prognosis, but this has not been elucidated in ovarian cancers. This study explores the potential difference in ploidy status between BRCA1-mutated and sporadic ovarian carcinomas. It also explores the potential association between BRCA1 mutation site and DNA ploidy status. Methods: This study compared DNA ploidy status of tumor blocks from 23 BRCA1-mutated ovarian carcinomas with that of 23 sporadic ovarian carcinomas matched for histologic subtype, patient age, stage and grade. DNA content of the nuclei was measured by Feulgen–Schiff staining followed by image cytometry and compared. Results: BRCA1-linked tumors with a stop codon closer to the N-terminal (between 1 and 500 aa; 6/6, 100%) had a significantly higher frequency of nondiploidy compared with those with stop codon above 500 aa (7/12, 58%) (P = 0.033). A diploid peak was detected in 28% of BRCA1-mutated ovarian cancers and in 33% of sporadic ovarian cancers. Conclusions: The present study concluded that ovarian tumors with mutations closer to the N-terminal of BRCA1 may have a higher risk of DNA aneuploidy. There is no significant difference between BRCA1mutated and sporadic ovarian carcinomas with respect to the DNA content. Key words: aneuploidy, BRCA1, DNA content, image cytometry, ovarian cancer, ploidy.
INTRODUCTION Previous studies have suggested a link between centrosome duplication, centrosome amplification defects, aggressive tumors and aneuploidy. BRCA1 is a tumor suppressor gene with a critical role in double-stranded DNA damage repair, which results in genomic stability. BRCA1 and p53, apart from their main function as tumor suppressor genes, play a significant role in con-
Correspondence: Prof Morteza Aghmesheh, MBBS, PhD, Department of Medical Oncology, The Wollongong Hospital, Crown Street, Wollongong, NSW 2500, Australia. Email: [email protected] Conflict of interest: The authors declare that there are no conflicts of interest Accepted for publication 7 September 2014.
© 2014 Wiley Publishing Asia Pty Ltd
trolling centrosome duplication by controlling the mitotic spindle assembly.1–4 Inhibition of BRCA1 function in mammary epithelial cells is acutely followed by DNA aneuploidy in these cells.5 BRCA1 localizes to centrosome along with other proteins that regulate the G2-M check point, including p53 and retinoblastoma (RB).6,7 Xu et al. showed in vitro that deletion of exon 11 of BRCA1 in fibroblast cells leads to unequal chromosome segregation, abnormal nuclear division and aneuploidy.1 Similarly, BRCA1-deficient cells obtained from knockout mouse embryos develop extra centrosomes and aneuploidy,1,8 suggesting that BRCA1 may play a role in maintaining normal DNA content during cell division, and as a corollary to this, that mutations in BRCA1 may lead to aneuploidy. Mutations in BRCA1 are associated with increased incidence of breast, fallopian and ovarian cancer.9–12 BRCA1 mutation-induced
2
genomic instability may facilitate the accumulation of multiple gene mutations.13 This could lead to activation of multiple signal pathways that play key roles in carcinogenesis, metastasis, prognosis and drug resistance.14–19 Consistently, BRCA1-linked breast cancers are more aneuploid compared with sporadic breast cancers20,21 and have a worse prognosis.22 Ploidy has been reported to be of prognostic significance in ovarian cancer and patients with diploid tumors have a significantly better survival than those with aneuploid tumors.23,24 There have been only few reports and only on small number of patients on either studies of ploidy in ovarian cancers from women with germline mutations in BRCA1 or comparing the DNA content of ovarian cancers in such patients with that of sporadic ovarian cancers. The aim of this study was to compare the DNA content in ovarian cancers from women with germline mutations in BRCA1 with sporadic controls to ascertain whether there were differences, and if so, whether there was any correlation with biological behavior.
METHODS Sample collection Paraffin-embedded ovarian tumors from 23 confirmed BRCA1-mutated ovarian cancer patients were collected from different hospitals in Sydney and KConFab. This group of patients was matched for histologic subtype, patient age, stage and grade with 23 ovarian cancer specimens from patients with no significant familial history of breast or ovarian carcinoma, termed sporadic cases.
Measurement of DNA content The tissue sections with more than 80% tumor tissue were used for the preparation of nuclei suspension to avoid contamination with normal tissue. Monolayers were prepared from 1 to 3 of 50-μm section/s using an established method.25 The DNA content of the nuclei was stained using Feulgen–Schiff staining procedure after monolayer preparation of paraffin sections. A Fairfield DNA ploidy system version 1.4.1 (Fairfield Imaging Ltd, Nottingham, UK) was used to process the images, which were stored with 1024 gray levels. The integrated optical density of each nucleus was calculated on the basis of measurements of optical density and area. Background optical density was measured and corrected for each nucleus.
© 2014 Wiley Publishing Asia Pty Ltd
M Aghmesheh et al.
At least 1500 nuclei were collected into galleries: lymphocytes, plasma cells and fibroblast nuclei were selected as reference cells. Cut, overlapped and pyknotic nuclei were excluded from the study. DNA histograms for each case were generated by image analysis. The resulting DNA histograms were classified based on their ploidy status (see the following section).
Classification of DNA ploidy The tumor was defined as diploid if only one G0/G1 peak (2c) was present in the histogram, the number of nuclei in the G2 peak (4c) did not exceed 10% of the total number of nuclei and the number of nuclei with a DNA content > 5c did not exceed 1%. A tumor was defined as tetraploid when a G0/G1 peak in the diploid position (4c) was present along with a G2 peak in the octa-position (8c, 8 copies of DNA) or the fraction of nuclei in the tetraploid region exceeded 10% of the total number of nuclei. The presence of a peak in the 8c position together with a G2 peak in the 16c position was an indication of polyploidy for a tumor.25 The tumor was defined as aneuploid when noneuploid peaks were present or the number of nuclei with DNA content > 5c, not representing euploid peaks, exceeded 1%. The histograms related to the DNA content were classified blindly (to the mutation status and clinical data) by Dr. Havard Danielsen. The coefficient of variation (CV) was calculated from the major peak in each case and the CV of the DNA diploid stem line in paraffin-embedded tissues was considered if it was 0.05).
Association of DNA content with stage, grade, histological subtype and patient’s age The majority of cases examined in this study were serous cystadenocarcinoma (93%, 39/42) and there were 2 Table 1 Distribution of DNA ploidy status in 18 BRCA1mutated and 23 sporadic ovarian carcinomas measured by DNA image cytometry
Diploid Tetraploid Aneuploid
BRCA1-mutated cases (%)
Sporadic cases (%)
5/18a (28) 3/18c (17) 10/18e (56)
7/23b (30) 3/23d (13) 13/23f (57)
Fisher’s test, P = 0.565 between a and b; P = 0.779 between c and d; and P = 0.599 between e and f.
Asia-Pac J Clin Oncol 2014
cases of mixed Mullerian tumor and two mixed tumors (serous + endometrial and serous + clear cell carcinomas). A summary of the association of aneuploidy DNA with age, stage and grade in BRCA1-linked and sporadic ovarian carcinomas is provided in Table 2. Of the entire study groups, observed DNA aneuploidy was numerically more common in subjects who were ≥50 years of age (67%, 12/18) compared with those below the age of 50 (48%, 11/23). However, this was not statistically significant (Table 2). There was more DNA aneuploidy in stage III (63%) and stage IV (60%) compared with stage II (17%) ovarian tumors. The difference between stage III and stage II reached statistical significance (19/30 vs 1/6, Fisher’s test, P = 0.043). No significant difference in DNA ploidy was observed among tumors of different histological grades (Table 2).
Association between BRCA1 and nuclear DNA content The association between BRCA1 mutation site and DNA content was investigated in 18 BRCA1-mutated ovarian cancers and their matched sporadic counterparts (Table 3). For statistical analysis, BRCA1-linked cases were divided into four groups according to their mutation site: BRCA1 mutations with stop codon less than 500 (amino acids), those with stop codon between 501 and 1000, stop codons between 1001 and 1500, and finally stop codons above 1500 (Fig. 1). BRCA1 mutation site was marginally correlated with DNA ploidy with more
Table 2 Association of aneuploid DNA with stage and grade in BRCA1-linked and sporadic ovarian carcinomas Number of aneuploid cases
Age (years) A 1294–1333 del 40 1471 C > A 1876 del C 3458 T > G 3450–3453 del CAAG 3450–3453 del CAAG 3875–3878 del GTCT 3875–3878 del GTCT 4184–4187 del TCAA 4184–4187 del TCAA 4184–4187 del TCAA Exon13 duplication Exon13 duplication 5382–5383 ins C
A A A T A A D D A D T A T A D A A D
BRCA1 mutations are ranked from N-terminal (top of table) toward the C-terminal (bottom). A, aneuploid; D, diploid; T, tetraploid.
Figure 1 Potential association between BRCA1 mutation site and DNA ploidy. Schematic view of BRCA1 protein with its N-terminal and C-terminal domains is demonstrated. DNA ploidy status of BRCA1-mutated ovarian cancers with stop codons ranging between 39 and 1829 is illustrated in this schematic view by circle (aneuploid), square (diploid) and triangle (tetraploid). Amino acids 504–803 of BRCA1 protein have been suggested to be a binding site for γ-tubulin with a potential role in DNA ploidy.
aneuploidy in cases having a mutation close to the N-terminal (with small amino acid number), while there were more euploid cases in those with a mutation close to the C-terminal (Kruskal–Wallis test, P = 0.050). Furthermore, a significant difference was noticed between two groups, aneuploid and non-aneuploid (diploid plus tetraploid), when analyzed using the Mann–Whitney U-test (P = 0.029). As indicated in Table 3, there are more aneuploid cases in patients with a stop codon close to the N-terminal of BRCA1 compared with those close to the C-terminal. Amino acids 504–803 of BRCA1 protein
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have been suggested to be a binding site with a potential role in DNA ploidy.27 Since the postulated binding site was between 504 and 803, for statistical purposes, BRCA1-mutated ovarian cancers were divided into two categories: cases with stop codons between 1 and 500 (close to the N-terminal of BRCA1 and prior to potential binding site) versus those with stop codons between amino acids 501 and 1863 (including the binding site) (Fig. 1). It was found that the BRCA1-linked tumors with a stop codon between 1 and 500 aa (6/6, 100%) had a significantly higher frequency of nondiploidy than those
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BRCA1 mutation and DNA content in ovarian cancer
with stop codon above 500 aa (7/12, 58%) (mean ranks: 14.42 vs 8.82; Mann–Whitney U-test, P = 0.033). As Table 3 demonstrates, tumor specimens with identical mutations demonstrated differences in DNA ploidy status.
DISCUSSION We are the first to report that there is significant correlation between BRCA1 mutation site and DNA aneuploidy in ovarian cancer samples. Of note, a specific domain of the BRCA1 protein, amino acids 504–803, was previously reported to be a binding site for γ-tubulin, an essential component of the centrosome.27 This encouraged us to divide BRCA1 mutations into two categories: (i) ovarian cancers with BRCA1 mutations resulting in a stop codon prior to 500 aa: such cases have lost the BRCA1 interaction site for γ-tubulin (amino acids 504–803) and (ii) those with stop codons above 500 aa, which may have lost the function of the interaction site for γ-tubulin. All BRCA1-linked ovarian cancers with a stop codon prior to 500 aa were nondiploid, whereas those with a stop codon above 500 were either diploid or tetraploid/aneuploid. This difference was statistically significant and is consistent with the data described by Hsu and White, suggesting that BRCA1 may be one of the key proteins in maintaining normal DNA content and that amino acids 504–803 provide a potential interaction site for this function of BRCA1.7 Further studies may focus on whether BRCA1related ovarian cancers with stop codons prior to 500 aa have poorer prognosis due to an increased chance of developing aneuploid tumors. What is the key factor involved in DNA aneuploidy of sporadic ovarian cancers if BRCA1 mutation has a role in DNA ploidy of BRCA1-related ovarian cancer? Somatic mutations in the p53 gene are another mechanism suspected of being involved in DNA ploidy status. We did not, however, find any significant association between p53 and DNA ploidy in either BRCA1-related or sporadic ovarian carcinomas in the present study. This is consistent with a previous study by Valverde et al. who found no association between DNA ploidy and p53.28 The other possible mechanism for DNA aneuploidy in sporadic ovarian cancers is BRCA1 inactivation through mechanisms other than mutation such as methylation.29 This is in support of the hypothesis that BRCA1-mutated ovarian cancers and at least a subpopulation of sporadic ovarian cancers develop through similar carcinogenic pathway.
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No published study has compared the DNA content in matched BRCA1-mutated and sporadic ovarian carcinomas. Our results indicated that there was no significant difference in the DNA content between the small size cohorts of BRCA1-mutated and sporadic ovarian carcinomas. This differs from the findings in BRCA1mutated breast cancers, which have a higher frequency of DNA aneuploidy and poorer prognosis compared with sporadic tumors.20–22 The results of the present study are in agreement with that of Auranen et al., who compared 58 familial ovarian cancer cases (not characterized by genetic testing in the patients) with 532 sporadic ovarian tumors for DNA ploidy by flow cytometry.30 These authors reported 46% aneuploid tumors in the familial group with no significant difference between familial and sporadic cancers. The advantages of the present study compared with Auranen et al.’s study are that ovarian tumor tissues with known BRCA1 mutation status were used in the present study and BRCA1-mutated tumors with their sporadic counterparts were matched for factors with potential influence on DNA ploidy status, namely histologic subtype, patient age, stage and grade.26 In addition, image cytometry used in this study is a more sensitive method of detecting aneuploidy than the flow cytometry used in Auranen et al.’s study.31,32 The lack of difference in DNA ploidy between BRCA1-linked and sporadic ovarian carcinomas may indicate that their carcinogenic pathways are similar. However, the potential “contamination” of the sporadic group in the present study may be a contributing factor to the absence of difference in DNA content between the two groups. BRCA1 mutations in the sporadic cohort of the present study was not tested due to ethical and practical reasons; data from other studies suggest that at least 10% of the sporadic cancers in the present study may have BRCA1 germline mutations.33 The possibility of a type II error (false-negative results due to a small sample size) exists and a study of larger cohorts is required to obtain more definite conclusion since the sample size in the present study is limited. DNA aneuploidy was more common in stage III compared with stage II ovarian cancers and that was statistically significant. There was no detectable difference between stage III and stage IV, possibly because of the limited number of stage IV ovarian cancers in the present study, but it may also indicate that aneuploidy is more important in the progression from stage II to stage III and less important from stage III to stage IV. This result suggests that DNA aneuploidy is more common in advanced ovarian cancers compared with early stages,
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which is in agreement with findings from results from a previous study.26 There was no significant correlation between grade and DNA aneuploidy or between age and aneuploidy in this study. In contrast, Trope and colleagues showed that aneuploid ovarian tumors are more often stage Ic (rather than Ia), poorly differentiated, of serous type, and in patients older than 50 years.26 As the majority of cases in the present study were serous, stage 3, grade 3 and in a younger age group in comparison with previously reported studies of sporadic ovarian cancers, the possibility of detecting any potential correlation between age, stage, histologic subtype or grade, and DNA ploidy was reduced. This study was limited by the sample size. Study of a larger cohort is required to confirm this result. The survival data were not accessible for most of the BRCA1- mutated group, limiting the ability to investigate any association between DNA aneuploidy and prognosis/response to chemotherapy, as well as links between the BRCA1 mutation site and prognosis/ response to chemotherapy. The sporadic group in the present study could not be tested for BRCA1 mutations because of ethical concerns. In conclusion, the present study is the first to demonstrate that ovarian tumors with mutations closer to the N-terminal of BRCA1 have a higher risk of DNA aneuploidy. It is tempting to postulate that BRCA1mutated ovarian carcinomas whose BRCA1 mutations result in a stop codon prior to 500 aa may behave differently in their prognosis; however, this needs to be formally tested in a larger prospectively collected sample. This study also found for the first time that there is no significant difference between BRCA1mutated and sporadic ovarian carcinomas with respect to the DNA content. This is in contrast to ploidy data in breast cancer where BRCA1-linked tumors were shown to be more aneuploid compared with their sporadic counterparts.
ACKNOWLEDGMENTS This study was supported by substantial donations from Mr. Steve Eckowitz and the Farmoz Company and in part by the National Institutes of Health (USA) (NIH CA 18119). KConFab has been funded by the Kathleen Cuningham Foundation, National Breast Cancer Foundation, National Health and Medical Research Council, Anti-Cancer Council of Victoria, Anti-Cancer Foundation of South Australia, Cancer Foundation of Western
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M Aghmesheh et al.
Australia and Queensland Cancer Fund and NSW Cancer Council.
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