In this study we aimed to address two questions; 1) what is the frequency of MMR inactivation in ovarian cancer, and 2) is it associated with platinum-based chemotherapy response.
First we analyzed eight ovarian cancer cell lines, i.e. SKOV6, HOC7, SKOV3, 2774, OVCAR3, KB3.1, CAOV3 and A2780. Microsatellite instability (MSI), which is a marker for MMR inactivation, was detected in three out of eight cell lines i.e. SKOV3, 2774 and A2780. This results in a frequency of MMR inactivation in ovarian cancer cell lines of 38%. The MSI in SKOV3 can be explained by the loss of MLH1 mRNA expression which, however, was not caused by promoter methylation. This is in agreement with the loss of MLH1 protein expression seen in SKOV3 described in a study of the 60 NCI cancer cell lines [44
]. In concordance with our findings, 2774 was also described to be MSI [45
]. One of the MSI positive A2780 sublines showed a strong methylation of the MLH1 promoter without MLH1 mRNA expression, while the other subline showed a low level of methylation and relative high mRNA expression. Strathdee et al. described that one MLH1 allele was methylated in A2780 [12
] which is comparable with the methylation status we saw in A2780, moreover one of our A2780 sublines showed complete methylation. On the other hand, another study did not detect MSI in A2780 [11
]. Interestingly, Aquilina and colleagues suggested there is a subpopulation of A2780 cells, estimated to be around one per 106
], which are MLH1 deficient and heterozygous for the p53phe172 mutation [46
]. Since these cells have a growth advantage, prolonged culturing of the A2780 cell line can result in selection of this subpopulation. Thus over time, separately cultured A2780 can have varying percentages of cells belonging to this subpopulation which may explain the discrepancies in MMR status seen in the A2780 cell lines analyzed by us.
Next we studied the association between MMR inactivation and cisplatin resistance in these cell lines. MMR inactivation seen in SKOV3 and 2774 might result in the relative resistance to cisplatin compared to the other cell lines. On the other hand, A2780 which has clearly an inactive MMR, was most sensitive to cisplatin. Overall, there seems to be no association between the response to cisplatin and MMR status in these eight cell lines. This is similar to a study in the 60 NCI cell lines which also showed no association between response to cisplatin and MMR status based on the MLH1 and/or MSH2 protein expression [44
Furthermore, we analyzed MMR status in 75 ovarian carcinomas to determine the frequency of MMR inactivation in ovarian cancer in vivo. Seven of the 75 ovarian carcinomas showed MLH1 promoter methylation. We confirmed whether the observed MLH1 promoter methylation results in the inactivation of the gene by determining the MLH1 mRNA expression with quantitative RT-PCR. The six tumors with low level MLH1 promoter methylation appeared to express MLH1 at mRNA levels similar to that of the unmethylated tumors. Thus a low level of methylation does not result in an altered expression of the MLH1 gene. In contrast, the abundant methylation seen in the remaining carcinoma was associated with the lowest MLH1 mRNA expression level of all 50 ovarian carcinomas tested. However, none of the ovarian carcinomas showed MSI for BAT25, BAT26 and for BAT40 or D2S123 which suggests a frequency of MMR inactivation of 0%. The low MLH1 mRNA expression seen in the abundant methylated carcinoma might be sufficient enough for a functional MMR which results in the observed absence of MSI.
Since ovarian cancer is a heterogeneous disease characterized by various histological types which may have different MSI frequencies, the number of specimens analyzed is very important in characterizing a feature that may be uncommon such as MSI. We therefore, made a summary of 20 studies totaling 1315 ovarian carcinomas, to compare the findings of these studies with our results (Table ). The MSI frequencies determined in these studies ranged from 0% to 39%. Overall, MSI was detected in 165 of the 1315 ovarian carcinomas tested, suggesting an overall incidence of 13% [18
Summary of the literature: Frequency of MSI in ovarian cancer. The total number of MSI, the number of MS markers used for the analysis and the number of MSI per histological subtype (if mentioned) is given for each study.
Multiple differences between these studies could have caused the wide range in the MSI frequency (0–39%). One of these is the number and variety of microsatellite markers analyzed to determine the MSI. The NCI recommended five markers comprising the National Cancer Institute Consensus Panel (NCI-CP) for the detection of MSI, i.e. markers for the mononucleotide repeats BAT25 and BAT26 and the dinucleotide repeats D2S123, D5S346 and D17S250 [48
]. Table shows per study the number of MS markers used and specifies how many of these are part of the NCI-CP. Interestingly, the studies that used all NCI-CP markers to determine the MS status also showed a wide range in MSI frequency (8–39%) which is similar to the overall range (0–39%). Therefore, the various MS markers used cannot be the sole cause for the wide range. Moreover, Gras et al. suggest that the reliability of the mononucleotide markers BAT25 and BAT26 is so high that most MSI can be predicted by evaluating these two markers exclusively [27
], confirming the less stringent role for the various markers used for the analysis.
Another difference between the studies is the distribution of the various histological types of the ovarian carcinoma tissues analyzed (Table ). This difference in the distribution could be a cause for the wide range in the MSI frequency especially since it has been suggested that certain histological types have a higher frequency of MSI. To determine whether there is a relation between histology and MSI within these studies, we looked at the frequency of MSI per histological type for the 628 patients with known histology (Table ). The summary of these studies suggests that the frequency of MSI is higher in the mucinous and endometrioid adenocarcinoma compared to clear cell and serous adenocarcinoma (the overall frequencies of MSI were 22%, 16%, 9% and 8%, respectively) (Table ). We hypothesize that mucinous and endometrioid histology might be prone to a higher MSI frequency since sporadic endometrial carcinoma, which is closely related to endometrioid ovarian cancer, has a MSI frequency of 20–30% [49
] and MSI is almost universal present in the colorectal tumors of the hereditary nonpolyposis colon cancer (HNPCC) syndrome which all have a mucinous histological type. Therefore, the different histology's of the ovarian carcinomas included in the several studies seems to be a plausible cause for the wide range in MSI frequency reported in these studies.
Next we addressed the second part of the aim of this study, is MMR inactivation associated with resistance to platinum-based chemotherapy in ovarian cancer. Forty-six of the 75 ovarian carcinomas we analyzed had been treated with platinum-based chemotherapy, eleven did not respond and 34 did. For one patient the response was not known. Methylation of the MLH1 promoter was detected in two of the eleven non-responders (18%) and four of the 34 responders (12%) and this was not significantly different (p = 0.664). Since we did not detect any MSI, the resistance seen in the eleven patients could not be associated with MSI and MMR inactivation.
The relation between MMR deficiency and platinum-drug resistance has been investigated in only a few in vivo
studies. Similarly to our result, no MSI was detected by Mesquita et al. [18
] who studied 34 ovarian carcinomas of which seven did not respond to cisplatin/paclitaxel therapy. So the resistance seen in these seven nonresponding patients was also not associated with MMR inactivation. In contrast, Samimi et al. [52
] found an inverse relation between MLH1 protein expression and the response to platinum-based chemotherapy in 54 ovarian carcinomas. Again, the number of ovarian carcinomas included in these studies is small and no further conclusion can be drawn from these results.
Since platinum-drug resistance is thought to be multifactorial the involvement of other resistance mechanisms could have overruled the possible contribution of MMR status. However, platinum treatment does seem to select for MMR deficient cells since in vitro
enrichment for MLH1 deficient colon cancer HCT116 cells in a mixed cell population was seen after cisplatin treatment [53
]. In addition, several in vivo
studies found an increase in the percentage of MSI and MLH1 methylation after platinum-based chemotherapy as well as a decrease in the percentage of cells positive for MLH1 and MSH2 [14
]. Moreover, an increase in MLH1 methylation after platinum-based chemotherapy was associated with poor survival in ovarian cancer patients [19
]. These results as well as the in vitro
studies mentioned in the introduction, suggest that MMR inactivation causes a low level resistance to platinum-based chemotherapy which does not play a significant role in intrinsic resistance. However, due to selection during chemotherapy MMR inactivation might play a greater role in the acquired resistance. We therefore propose that the role of MMR inactivation in acquired resistance in ovarian cancer should be further investigated.