Evaluation of DNA damage by Alkaline Comet Assay in early breast cancer patients

F.
1, Galardi

M.

1, Truglia

S.

1, Cappadona

S. Bessi, S.

1, Licitra

C.

1, Oakman

L.

1, Biganzoli

L.

2, Grisotto

D.

2, A. Catelan

2, Biggeri

1 A.Giannini

and A. Di

1 Leo

1 Translational Research Unit Department of Oncology Hospital of Prato, 2 Department of Statistics "G. Parenti" University of Florence and Biostatistics Unit, ISPO, Florence
INTRODUCTION
Breast cancer comprises an extraordinarily diverse group of diseases in terms of presentation, morphology, molecular profile and treatment response. Gene expression analysis identifies four main breast cancer subgroups that are biologically and clinically distinct (1). The Triple-negative (TN) subgroup shows an unfavourable prognosis with aggressive behaviour and few effective treatment strategies. Expression of basal markers identifies a biologically and clinically distinct subgroup of TN tumors (Basal like, BL). BL tumours have a dysfunctional BRCA1 pathway resulting in a particular sensitivity to DNA damaging agents (2). Alkaline comet assay is broadly accepted as a standard method for assessing DNA damage and DNA repair ability in individual cells (4). It provides valuable information about intrinsic DNA characteristic and responses to external factors, including radiation, chemicals and drugs. This information may prove particularly relevant in the diagnosis, prognosis and treatment of cancer (5). Our aim was to determine if a correlation exists between breast cancer molecular sub-groups and DNA damage and to use and validate Comet assay to identify patients with a cancer defective in DNA repair machinery.
Tab.1 DNA DAMAGE
DNA damage markers Sub-groups LA n=62 LB n=19 Her2+ n=8 TN n=15 Sub-group TN TN BLn=7 BL n=8 0.56 0.52 0.37 0.58 0.61 0.58 0.41 0.63 1.10 0.89 0.99 1.03 1.18 0.98 1.04 1.19 log Relative Mean TI mean 0.33 0.54 0.62 0.54 sd 0.27 0.64 0.37 0.47 log Relative Median TI mean * 0.36 0.58 0.73 0.60 sd 0.33 0.38 0.44 0.52 log Relative Mean TM mean 0.54 0.96 0.78 0.99 sd 0.49 0.74 0.53 0.98 log Relative Median TM MEAN * 0.59 0.95 0.93 1.07 sd 0.57 0.79 0.69 1.08

Fig. 3 Box plot

Tab.1 Mean and standard deviation of DNA damage markers.

* markers median values were mediated in order to apply ANOVA test.

Tab.2 DNA FRAGMENTATION FREQUENCES

Sub-groups DNA damage markers % of DNA fragmentation Quintiles % 0-20 LA n=62 n 17 16 10 14 5 16 16 11 14 5 16 15 11 12 8 16 13 13 12 8 % 27.42 25.81 16.13 22.58 8.06 25.81 25.81 17.74 22.58 8.06 25.81 24.19 17.74 19.35 12.90 25.81 20.97 20.97 19.35 12.90 n 1 2 6 5 5 1 3 6 4 5 1 2 5 7 4 2 4 2 7 4 LB n=19 % 5.26 10.53 31.58 26.32 26.32 5.26 15.79 31.58 21.05 26.32 5.26 10.53 26.32 36.84 21.05 10.53 21.05 10.53 36.84 21.05 Her2+ n=8 n 0 1 2 2 3 0 1 2 2 5 0 2 3 1 2 0 1 5 0 2 % 0.00 12.50 25.00 25.00 37.50 0.00 12.50 25.00 25.00 37.50 0.00 25.00 37.50 12.50 25 0.00 12.50 62.50 0.00 25.00 n 2 2 3 0 8 3 1 2 1 8 3 2 2 1 7 2 3 1 2 7 TN n=15 % 13.33 13.33 20 0.00 53.33 20.00 6.67 13.33 6.67 53.33 20.0 13.33 13.33 6.67 46.67 13.33 20.00 6.67 13.33 46.67

Sub-group TN TN BLn=7 n 0 % 0.00 n BL n=8 % 0.00 0.00 2 25.00

Fig. 1a, Healthy cells

Fig. 1b, Cancer cells

Fig. 2a) Healthy Cells Fig. 2b,c,d,e) Cancer Cells

log Relative Mean TI

20-40 40-60 60-80 80-100 0-20 20-40 40-60 60-80 80-100 0-20 20-40 40-60 60-80 80-100 0-20 20-40 40-60 60-80 80-100

2 28.57 0 0 0.00 0

2 28.57 1 12.50 3 42.86 5 62.50 1 14.29 2 25.00 1 14.29 0 1 14.29 0 0.00 0.00 1 14.29 1 12.50 3 42.86 5 62.50 1 14.29 2 25.00 2 28.57 0 0 0.00 1 14.29 0 0 0 0.00 0.00 0.00 0.00 2 25.00

MATERIALS AND METHODS

Scrapings from 104 primary BCs and respective healthy tissues were collected for Alkaline Comet Assays (Trevigen). The presence of healthy and cancers cells was confirmed by pathologist review of Papanicolau slides (Fig 1). Using IHC (ER, PGR, HER2, Ki67, CK5/6) and grade, samples were classified as Luminal A (LA) (n=62), Luminal B (LB) (n=19), HER2+ (n=8), Triple negative (TN) (n=15). The TN sub-group WAS classified as Basal like (BL) (n=7) and TN without Basal Like (TN BL-) (n=8). DNA damage (COMET IV software, Perceptive), was measured as Tail Intensity (TI), directly related to DNA break frequency, and Tail Moment (TM), a combination of tail length and TI. To analyze the differences between subgroups, 4 estimators were used: Log relative mean TI: log [(mean TI cancer cells /mean TI healthy cells)], Log relative median TI: log [(median TI cancer cells /median TI healthy cells)], Log relative mean TM: log [(mean TM cancer cells /mean TM healthy cells)], Log relative median TM: log [(median TM cancer cells /median TM healthy cells)]. STATISTICAL ANALYSIS ANOVA, Bartletts test and Variance Ratio test were used to analyse the difference between groups.

log Relative Median TI

log Relative Mean TM

3 42.86 4 50.00 2 25.00 0.00 1 12.50 3 42.86 0

log Relative Median TM

1 14.29 1 12.50 3 42.86 4 50.00

Tab.2 Number of breast cancer cases and row percentage by quintiles of DNA damage markers.

Fig. 3 Box plot of DNA damage markers by breast cancer sub-group.

RESULTS
Sub-group means (Tab.1) were different considering Log Relative Mean TI, Log Relative Median TI and Log Relative Mean TM (Anova test, p=0.008, p=0.01 and p=0.02 respectively). Significant differences were found between subgroup variances (Bartletts test, Log Relative Mean TI p=0.03, Log Relative Mean TM p=0.003, Log Relative Median TM p=0.008). The largest difference was between the variance of TN subgroup and the other subgroups variance (Log Relative Mean TI p=0.02, Log Relative Mean TM p=0.003, Log Relative Median TI p=0.003). In Tab. 2 is reported the sub-group distribution of DNA fragmentation markers. Luminal A subgroup distribution was concentrated on the lowest fragmentation percentiles (<40 percentile), Luminal B distribution was more dispersed, Her2+ distribution was concentrated above the 60 percentile. TN population show high levels of fragmentation (about 50% of patiens in the highest quintile of DNA fragmentation markers). Box plots of TI and TM markers show that TN sub-group had the highest DNA fragmentation level and the largest variability. Luminal A sub-group showed the lowest DNA fragmentation level. Within TN sub-group, BL population was not significantly different from TN BL- one, either considering DNA fragmentation level either regarding population variability.

CONCLUSION
We found an association between breast cancer molecular sub-groups and DNA damage evaluated by Comet Assay as Tail Intensity and Tail Moment. In particular, our results show differences between sub-groups in terms of average DNA damage. Around 50% of Luminal A cancer shows low DNA damage (less than 40 percentile of fragmentation) while more than 60% of HER2+, TN and BL cancers show high levels of DNA fragmentation (gretaer than 60 percentile of fragmentation). We found a large between-patients variability within sub-groups. In particular the Triple Negative sub-group variance was almost twice the other sub-groups variance. THE BL sub-group seemed to reflect the same behaviour than THE TN sub-group but too few patients were analysed. This result highlights a large residual heterogeneity of these cancers, which need further research efforts. In conclusion, Comet Assay seems to be a sensitive tool able to identify patients with a defective DNA repair machinery. Further clinical validation IS necessary, moreover in vitro studies are ONGOING in order to EXPLORE THIS TEST AS AS A TOOL PREDICTING ACTIVITY OF DNA DAMAGING CYTOTOXICS
BIBLIOGRAPHY
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