We analyzed 9 clinical samples of recurrent BRCA1-mutated ovarian cancer previously treated with platinum (). Six of them were clinically platinum-resistant and 3 were platinum-sensitive. One case (UW40) showed primary resistance to platinum, while 5 cases were initially sensitive to platinum and acquired resistance during the disease course. Three of the 5 tumors with acquired platinum resistance (UW80, UW91 and UWF27) and the primarily resistant tumor (UW40) had secondary genetic changes in BRCA1, while none of the three cisplatin-sensitive recurrent tumors showed secondary genetic changes in BRCA1.
UW80 patient is a heterozygous carrier of a frameshift BRCA1
mutation, 185delAG, which is common in the Ashkenazi Jewish population (17
) (). In the primary tumor specimen, only BRCA1
sequence with 185delAG was detected, indicating that the tumor had lost the wild-type allele. Loss of heterozygosity (LOH) of an intragenic BRCA1
single nucleotide polymorphism (SNP) was also confirmed. In the microdissected recurrent tumor from the same patient after development of resistance to platinum, LOH of the BRCA1
SNP was confirmed, indicating that the recurrent tumor contained only the same single allele of BRCA1
as present in the primary tumor and that contamination of non-tumor cells was negligible. In this recurrent sample, wild-type BRCA1
sequence represented >80% of the sequences at the mutation site, suggesting that the recurrent tumor had acquired wild-type BRCA1
by genetic reversion (back mutation to wild-type).
Genetic reversion of BRCA1 mutation in 3 recurrent BRCA1-mutated ovarian cancer cases
Similarly, in 2 more BRCA1.185delAG
cases (UW91 and UWF27), the primary tumor demonstrated only mutant sequence with loss of the wild-type allele. In the recurrent tumors, we mainly detected wild-type BRCA1
sequence, suggesting that these two platinum-resistant tumors acquired wild-type BRCA1
by genetic reversion (back mutation to wild-type) (). Both cases had confirmation of LOH at intragenic SNPs in the primary and recurrent tumors (Supplemental Fig. 1
), indicating the presence of a single BRCA1
allele that underwent a secondary genetic alteration.
BRCA1.185delAG encodes a severely truncated protein with 38 amino acids, and the back mutation should restore full length BRCA1 protein (). Consistently, immunohistochemical study revealed that BRCA1 protein expression was absent in the primary tumors of UW80 and UW91, but strongly positive in their recurrent tumors ( and data not shown).
BRCA1 protein expression is restored in recurrent ovarian tumors with genetic reversion of BRCA1 mutation
The mechanism for genetic reversion of 185delAG mutations in all three tumors is not clear. At the genomic level, the re-insertion of two missing nucleotides would seem less likely to occur than a secondary downstream deletion that corrects the frameshift and restores the open reading frame. Several possible explanations for this phenomenon are not mutually exclusive. First, we speculate that because 185delAG is in a critical portion of the BRCA1 gene close to the RING finger (), a change of one single amino acid here is likely to have deleterious effect on protein function. Similarly, missense mutations in the RING finger such as C61G can be deleterious (18
). Therefore restoration of BRCA1 function may require complete reversion of a mutant in this domain to wild-type. Another possibility is that the particular genomic sequence surrounding 185delAG somehow facilitates the genetic reversion. A third possibility is that other downstream mutations do occur in some tumors but are not detected with our relatively short PCR amplifications. We are unable to do long range PCR that might identify larger genomic events because of the fragmented nature of our DNA. Thus, it is easier to identify genetic reversion in our samples than larger events that restore the open reading frame.
UW40 patient is a heterozygous carrier of a frameshift BRCA1 mutation, 2594delC (). The mutant allele encodes an 844 amino acid protein that lacks BRCT domains (). The patient had a past history of breast cancer at the age of 42 and developed ovarian cancer at age 54. The primary ovarian carcinoma was clinically resistant to platinum. After 6 cycles of platinum and taxol, the patient was treated with taxol for 3 additional cycles with development of progression. She was re-treated with topotecan, then gemcitabine and adriamycin to obtain a partial remission. Recurrent tumor was then obtained at the time of surgery for a fistula.
Genetic reversion of BRCA1 mutation in a primary ovarian tumor with platinum resistance and another secondary BRCA1 mutation in a recurrent tumor of the same patient
LOH of three intragenic BRCA1 SNPs (2201C/T, 2430T/C, and 2731C/T) that flank the mutation site was confirmed in both the primary and recurrent tumor (, and data not shown), indicating that contamination by non-tumor cells was negligible and that both the primary and recurrent tumors had lost one BRCA1 allele. Intriguingly, in the primary tumor, both wild-type BRCA1 sequence and BRCA1 sequence with 2594delC were detected (). Careful laser microdissection of a separate second sample of this tumor revealed the same result. The presence of both wild-type BRCA1 sequence and mutant sequence on one allele in the primary tumor suggests that genetic reversion (back mutation to wild-type) occurred on one copy of the mutant allele. We speculate that the presence of the genetically-reverted wild-type allele in the primary tumor contributed to the unusual initial platinum resistance of this tumor. The selective pressure for the genetically reverted tumor cells in the primary ovarian tumor is unknown, but may be related to the 11 months of cyclophoshamide, methotrexate and fluorouracil that the patient previously received for breast cancer. Cyclophosphamide is a DNA cross-linking agent and could exert similar selection pressure for the correction of DNA repair defects as we have observed with exposure to platinum agents.
An alternative explanation for the existence of genetic reversion and other secondary mutations on a mutant allele in this primary tumor is that these events are more common than we appreciate but usually occur in only a small number of primary tumor cells before exposure to chemotherapy. Then, chemotherapy provides the selection pressure for expansion of these sub-clones in recurrent tumors. Perhaps some reports of retention of the wild-type BRCA1/2
allele in breast cancers (19
) actually represent genetic reversion. Only careful haplotyping distinguishes tumors with retention of the wild-type allele from those with genetic reversion.
The genetically-reverted wild-type allele was then lost at some point during the disease course. In the recurrent tumor, we could no longer detect wild-type sequence, but we found a second site small deletion (2606_2628del23) in addition to the inherited mutation (2594delC). The second mutation corrects the frameshift caused by the inherited mutation and the allele BRCA1.2594delC;2606
encodes a BRCA1 protein with intact C-terminal region, which may be functional (). The actual genomic DNA sequence of BRCA1
in the UW40 recurrent tumor is a mixture of 2594delC;2606_2628del23 and 2594delC without a secondary mutation (). The mixture of sequences on one allele in both the primary and recurrent tumor may have occurred by secondary genetic alterations (reversion to wild-type in the primary tumor and 23 bp deletion in the recurrent tumor) occurring on just one of the duplicated mutant alleles (), similar to the situation occurring in Capan-1 clones with secondary mutations of BRCA2
observed in the in vitro
experiment in the previous report (12
). Alternatively, the two sequences could represent tumor heterogeneity, with only a subset of tumor cells acquiring a secondary mutation on the mutant allele. We favor the explanation of tumor heterogeneity in this case, given that a cell line developed from this recurrent tumor contains only mutant sequence without secondary genetic changes (15
Taken together, secondary mutations that restore the reading frame of mutated BRCA1 alleles were frequently observed in ovarian tumors with resistance to platinum. These results suggest that secondary mutations of BRCA1 can be a mechanism of acquired resistance to cisplatin in BRCA1-mutated ovarian cancer. In addition, the observation of the back mutation of BRCA1 in cisplatin-resistant primary tumor of UW40 patient suggests that secondary mutations of BRCA1 can be a mechanism of primary resistance to cisplatin.
Our studies in this paper and in the previous paper (12
) emphasize that BRCA1
are not only cancer susceptibility genes, but are also critical determinants of clinical sensitivity and resistance to chemotherapy. These studies support the general concept that defective DNA repair leads to chemosensitivity, while restoration of functional DNA repair contributes to acquired chemoresistance in tumor cells in vitro
and in vivo
Testing for secondary mutations in platinum-treated BRCA1-mutated cancers may be clinically important, because tumors with secondary mutations are likely to be resistant to platinum and could demonstrate cross-resistance to other agents that exploit BRCA1 defects such as poly(ADP-ribose) polymerase 1 inhibitors. A larger clinical study is warranted to further determine the prevalence and clinical significance of secondary mutations of BRCA1 in BRCA1-mutated cancer.
Our finding could have implications for sporadic ovarian carcinoma. Although somatic mutation of BRCA1/2
is rare in sporadic ovarian carcinoma, BRCA1 is reported to be down-regulated in a subset of sporadic ovarian carcinomas by promoter methylation and other unknown mechanisms (20
mRNA expression is reported to be undetectable by reverse transcription PCR in 13% of ovarian carcinoma (20
). Therefore, it is tempting to hypothesize that down-regulation of BRCA1/2 causes initial sensitivity to cisplatin, and restoration of functional BRCA1/2 expression by demethylation of the BRCA1
promoter or other mechanisms leads to acquired resistance. Testing this hypothesis will be important to elucidate the more general roles of BRCA1/2 in platinum-sensitivity and resistance.