Clinicopathologic significance and race-specific prognostic association of MYB overexpression in ovarian cancer

Late diagnosis, unreliable prognostic assessment, and poorly-guided therapeutic planning result in dismal survival of ovarian cancer (OC) patients. Therefore, identifying novel functional biomarker(s) is highly desired for improved clinical management. MYB is an oncogenic transcription factor with emerging functional significance in OC. Here we examined its clinicopathologic significance by immunohistochemistry and TCGA/GTex data analyses. Aberrant MYB expression was detected in 94% of OC cases (n = 373), but not in the normal ovarian tissues (n = 23). MYB was overexpressed in all major epithelial OC histological subtypes exhibiting the highest incidence (~ 97%) and overall expression in serous and mucinous carcinomas. MYB expression correlated positively with tumor grades and stages. Moreover, MYB exhibited race-specific prognostic association. Moderate-to-high MYB levels were significantly associated with both poor overall- (p = 0.02) and progression-free (p = 0.02) survival in African American (AA), but not in the Caucasian American (CA) patients. Consistent with immunohistochemistry data, we observed significantly higher MYB transcripts in OC cases (n = 426) than normal ovary (n = 88). MYB transcripts were significantly higher in all epithelial OC subtypes, compared to normal, and its greater levels predicted poor survival in AA OC, but not CA OC, patients. Thus, MYB appears to be a useful clinical biomarker for prognostication, especially in AA patients.

www.nature.com/scientificreports/ therapy 8,10 . Significant molecular differences also exist among histological subtypes, which remain to be characterized fully for clinical exploitation in biomarker and therapeutic development.
MYB is a proto-oncogene that encodes for a transcription factor protein 11,12 . Defects in the chromosomal region harboring MYB were first reported in human acute myelogenous leukemia 12 . Since then, its overexpression and other abnormalities have been detected in several other malignancies [13][14][15][16][17] . MYB plays an essential role in maintaining the undifferentiated proliferative state of immature hematopoietic cells, and its knockout causes the loss of blood cell lineages in mice and is embryonically lethal 18,19 . Information on MYB functions in OC pathobiology has begun to emerge recently 20,21 , underscoring the need to study its expression pattern and clinicopathologic significance in OC.
In the present study, we investigated MYB expression in situ by immunohistochemistry in normal and malignant ovarian tumors. We also evaluated MYB mRNA expression in OC and its clinicopathologic correlation using publicly available transcriptomic databases. Our findings demonstrate significant MYB overexpression in all EOC histological subtypes, with the most pronounced expression detected in SC and MC. MYB at the protein level correlated positively with increasing tumor-grade and clinical stages. Moderate-to-high MYB protein levels also predicted worse progression-free survival. Although no significant difference in MYB expression was observed between AA and CA OC, high MYB expression was a stronger predictor of poor overall and progression-free survival in AA women. Thus, our study suggests that MYB can be a useful diagnostic and prognostic biomarker for ovarian malignancy, particularly in AA patients.
MYB overexpression is a predictor of poor disease outcomes, especially in African American ovarian cancer patients. Since MYB expression exhibited a positive association with increasing tumor grade and stage, we next examined if MYB levels correlated with the survival of OC patients by performing Kaplan-Meier analysis. Survival data were available for a total of 74 OC cases, of which 36 and 38 belonged to AA and CA women with OC, respectively. We first used a mean composite score cut-off value of ≤ 6 as low MYB and ˃6 as high MYB to examine a survival association. Our data demonstrated a trend of poor -overall and -progression-free survival in AA OC patients with high MYB expression; however, it failed to reach the statistical significance (Supplementary Figure S2). Next, we examined the survival association by keeping the mean composite score of ≤ 4 as a cut-off for the low MYB-expressing group, and cases with a composite score of > 4 were categorized as moderate to high MYB-expressing group. Based on this criterion, we observed a significantly worse progression-free survival (p = 0.04) of the OC patients with moderate/high MYB expression than those with low MYB expression. In addition, a clear trend towards poor overall survival of the OC patients with moderate to high MYB expression was also recorded compared to the low MYB group (p = 0.05) (Fig. 3A). Since women of AA racial background have greater mortality from OC despite having a lower incidence than the CA women 22 , we examined the prognostic association of MYB separately in these groups. We observed a significantly worst overall (p = 0.02) and progression-free (p = 0.02) survival of the AA subjects with moderate to high MYB expression when compared to the low MYB expressing group (Fig. 3B). However, we did not observe a significant change in overall and progression-free survival of the CA subjects with mod/high MYB expression compared to the low MYB expressing group (Fig. 3C). Thus, our data suggest that MYB could be a stronger predictor of survival in AA women with OC diagnosis. www.nature.com/scientificreports/

Expression pattern of MYB transcript and its prognostic association in OC patients. To further
generate support for our observations, we next analyzed the transcriptomic data available in multiple public databases, including GTEx (Genotype-Tissue Expression), TCGA-ovarian cancer project (The Cancer Genome Atlas), and individual laboratory studies. These databases were searched by using interactive analytical platforms, GEPIA, UALCAN, and ONCOMINE. The data mining revealed significantly higher expression of MYB transcript (false discovery rate-adjusted) in a large ovarian cancer cohort (n = 426) compared to the normal  www.nature.com/scientificreports/ ovarian tissues (n = 88) (Fig. 4A). A differential expression of MYB transcripts among histological OC subtypes was also recorded (Fig. 4B, C). No significant differences in MYB expression were recorded in AA and CA OC cases in the TCGA database (Fig. 4D). Similarly, no significant association of MYB transcript levels with clinical stages or histological grade was observed (Fig. 4E, F). MYB also did not significantly associate with overall or progression-free survival (Fig. 5A, B). When examined in AA and CA groups, a significant association of high MYB with lower survival was recorded in AA women with OC (p = 0.021) but not in CA women (p = 0.57) (Fig. 5C, D). These data suggest that MYB is aberrantly expressed at both RNA and protein levels in OC and could serve as a predictor of survival, especially in AA women with OC. Further, MYB should also be explored as a functional target associated with racially disparate clinical outcomes in AA women.

Discussion
The scarcity of reliable screening tools for early-stage diagnosis is one of the major challenges associated with the poor survival of OC patients 23,24 . Further, lack of clarity about the cell of origin and molecular and histological complexities make the therapeutic planning and prognostication of epithelial OC clinically challenging 6,10 . Identification of functional biomarkers associating with disease aggressiveness and clinical outcomes could allow for better therapeutic planning, disease monitoring, and the development of novel targeted therapeutics. www.nature.com/scientificreports/ Indeed, existing and emerging studies continue to demonstrate the strength of molecular markers in accurate histopathological and clinical diagnosis, prognosis, and treatment guidance. The present study identified MYB as a potentially useful clinical biomarker for OC. Like other transcription factors, activated MYB is localized in the nucleus, where it regulates the expression of genes associated with different cellular processes 11,17,25 . The earliest documentation of aberrant MYB expression in OC came from a study that examined MYB transcripts in OC cell lines and a few clinical cases 26 . Our study examined MYB expression in a larger cohort of normal and ovarian cancer cases and found its widespread overexpression in all EOC histological subtypes. Staining intensity varied (weak to strong) between and within cases suggestive of the heterogeneous disease or the cells in various progression stages. The variable expression of MYB in OC cases could also represent molecular heterogeneity resulting from genetic differences or epigenetic alterations in cell populations due to their exposure to the differing tumor microenvironment.
MYB expression was not detected in any of the normal ovary cases suggesting that its dysregulation occurs following malignant transformation, and thus it could be of potential diagnostic significance. As expected, MYB expression was predominantly nuclear, although diffuse staining in the cytoplasm was also detected. Therefore, it will be interesting to pursue if MYB also has any non-nuclear functions besides gene regulation. Further, among all histological subtypes, SC and MC exhibited the highest expression. p53 mutations have been reported in both these subtypes 10 . Thus, molecular crosstalk between MYB and p53 can aid SC and MC, requiring further validation in preclinical and clinical settings.
Irrespective of the histological subtypes, MYB protein expression positively correlated with OC's progressive histological grades and clinical stages. This is in line with an established role of MYB in cellular differentiation and promotion of malignant behavioral features 12,16,17,25,27 . It also underscores the need to delineate the molecular mechanisms involved in MYB dysregulation in OC to develop disease prevention and therapy strategies. Prediction of worst progression-free survival by the moderate-to-high MYB protein expression in OC cases further strengthens the notion that MYB plays an important role in disease aggressiveness. Moreover, stronger MYB association with poor survival in AA patients is also exciting and warrants further investigations. This is quite interesting and suggests that MYB likely acts in concert with other molecular factors in AA OC to impact tumor biology leading to racially disparate clinical outcomes. In fact, we have previously observed that increased expression of indoleamine 2.3-dioxygenase in AA OC patients associated with poor survival 28 . Thus, it will be interesting to examine a mechanistic link of MYB with other proteins exhibiting racially disparate expression in OC. It is also interesting to note that a lower cutoff of MYB expression levels provided a better correlation with www.nature.com/scientificreports/ survival outcomes. It suggests that MYB being a transcription factor can significantly impact the transcriptome and, as a result, cellular phenotypes upon slight increases in its expression. Aberrant expression of MYB was also apparent at the mRNA level, suggesting that its dysregulation in OC likely occurs at the genomic (gene amplification), transcriptional and/or post-transcriptional levels. Indeed, in other cancers, both gene amplification and transcriptional upregulation of MYB have been reported 11,14 . Further, post-transcriptional regulation via microRNAs is also documented 21,29 . Unlike protein, the MYB transcript did not significantly associate with histological grades and clinical stages due to the smaller sample size. A statistically significant association of higher MYB mRNA levels with poor survival outcomes in AA patients further supports its role in racial disparity requiring further investigations.
In conclusion, our study establishes the clinical significance of MYB in OC that should be explored further in combination with other biomarkers. Functional and in-depth mechanistic studies including identification of transcriptional targets of MYB in OC should also be pursued. These efforts will yield information that could be exploited to develop MYB-targeted therapeutic strategies and clinical utilization of MYB as a useful biomarker of diagnostic and prognostic significance. Immunohistochemistry. IHC was performed on formalin-fixed, paraffin-embedded normal or ovarian tumor tissue sections as previously described 25 . In brief, tissue sections were deparaffinized, hydrated, and subjected to antigen retrieval in Decloaking Chamber (Biocare Medical, Concord, CA). After that, peroxide  and scored for the nuclear staining of MYB on a three-point scale, 1+ (weak); 2+ (moderate); and 3+ (strong), and the percent positivity on a four-point scale, 0-25% ('1'), 26-50% ('2'), 51-75% ('3'), and 76-100% ('4') using standard scoring recommendation 30 . Weak (1+) staining in less than 10% of cells was considered negligible and given a value of '0' . A composite score was calculated for each section by multiplying the staining intensity and percent positivity values, ranging from 0 to 12 30 .
Besides, an association of MYB mRNA with race, clinical stages, histological grades, and patient survival was also studied.

Statistical analysis.
We employed Wilcoxon, Mann-Whitney, Anova, Chi-square, Fisher's exact tests, and logistic regression model as appropriate for all statistical data comparisons between various experimental groups. Overall survival curves were developed using the Kaplan-Meier method and compared using the stratified log-rank test. All p-values were two-sided, and all confidence intervals were at the 95% level. A p-value of < 0.05 was considered statistically significant. Computation for all the analyses was performed using the Statistical Analysis System (SAS).

Data availability
The data presented in this article is available for sharing upon a reasonable request.