Distinct Microbial Signatures Associated With Different Breast Cancer Types

Abstract

A dysbiotic microbiome can potentially contribute to the pathogenesis of many different diseases including cancer. Breast cancer is the second leading cause of cancer death in women. Thus, we investigated the diversity of the microbiome in the four major types of breast cancer: endocrine receptor (ER) positive, triple positive, Her2 positive and triple negative breast cancers. Using a whole genome and transcriptome amplification and a pan-pathogen microarray (PathoChip) strategy, we detected unique and common viral, bacterial, fungal and parasitic signatures for each of the breast cancer types. These were validated by PCR and Sanger sequencing. Hierarchical cluster analysis of the breast cancer samples, based on their detected microbial signatures, showed distinct patterns for the triple negative and triple positive samples, while the ER positive and Her2 positive samples shared similar microbial signatures. These signatures, unique or common to the different breast cancer types, provide a new line of investigation to gain further insights into prognosis, treatment strategies and clinical outcome, as well as better understanding of the role of the micro-organisms in the development and progression of breast cancer.

Keywords: HER2 positive breast cancer; endocrine receptor positive breast cancer; microbiome; triple negative breast cancer; triple positive breast cancer.

Figure 1

Viral signatures associated with different breast cancer types. Among the breast cancer types, the endocrine receptor (estrogen/progesterone) positives are abbreviated as BRER, human epidermal growth factor receptor 2 positives are abbreviated as BRHR, triple positives (estrogen, progesterone and HER2 receptor positive) are abbreviated as BRTP and the triple negatives (absence of estrogen, progesterone and HER2 receptors) are abbreviated as BRTN. The normal breast control samples obtained from healthy individuals are abbreviated as NC. (A) Venn diagram showing the common and unique viral signatures in the 4 types of breast cancers. (B) The heat map of common viral signatures in the 4 breast cancer types. (C) Relative hybridization signals of viral probes detected in breast cancer types. For example, hybridization signals for Polyomaviridae probes were 4, 6, and 3% of the total hybridization signals detected in BRER, BRTP, and BRHR respectively. (D) Prevalence of viral signatures in 4 breast cancer types. Since the hybridization signals for Polyomaviridae, Hepadnaviridae and Parapoxviridae were lower than the cut-off (log2 fold change in hybridization signal >1) in one or more breast cancer types they are depicted as negative in this figure. However, (E) shows the heat map of hybridization signals for those viral signatures to be still significantly higher in the cancers when compared to the control.

Figure 2

Bacterial signatures associated with different breast cancer types. Among the breast cancer types, the endocrine receptor (estrogen/progesterone) positives are abbreviated as BRER, human epidermal growth factor receptor 2 positives are abbreviated as BRHR, triple positives (estrogen, progesterone and HER2 receptor positive) are abbreviated as BRTP and the triple negatives (absence of estrogen, progesterone and HER2 receptors) are abbreviated as BRTN. The normal breast control samples obtained from healthy individuals are abbreviated as NC. (A) Bacterial phyla associated with breast cancer types. (B) Venn diagram showing the common and unique bacterial signatures in the 4 types of breast cancers (C). The heat map of common viral signatures in the 4 breast cancer types. (D) Hybridization signals of bacterial probes detected in breast cancer types. (E) Prevalence of bacterial signatures in 4 breast cancer types.

Figure 3

Fungal and parasitic signatures associated with different breast cancer types. Among the breast cancer types, the endocrine receptor (estrogen/progesterone) positives are abbreviated as BRER, human epidermal growth factor receptor 2 positives are abbreviated as BRHR, triple positives (estrogen, progesterone, and HER2 receptor positive) are abbreviated as BRTP and the triple negatives (absence of estrogen, progesterone, and HER2 receptors) are abbreviated as BRTN. The normal breast control samples obtained from healthy individuals are abbreviated as NC. (A) Relative hybridization signals of fungal probes detected in breast cancer types. For example hybridization signals for Ajellomyces were 7, 8, and 14% of the total hybridization signals detected in BRER, BRTP, and BRHR respectively, and that of Rhizomucor is 19% of the hybridization signals detected in BRTN. (B) Prevalence of viral signatures in 4 breast cancer types. (C) Venn diagram showing the common and unique fungal signatures in the 4 types of breast cancers. (D) Relative hybridization signals of parasitic probes detected in breast cancer types. For example hybridization signals for Plasmodium were 10, 6, and 21% of the total hybridization signals detected in BRER, BRTP, and BRHR respectively, and that of Mansonella is 7 and 12% of the hybridization signals detected in BRTP and BRTN respectively. (E) Prevalence of parasitic signatures in 4 breast cancer types. (F) Venn diagram showing the common and unique parasitic signatures in the 4 types of breast cancers.

Figure 4

Hierarchical clustering of BRER (A), BRTP (B), BRHR (C), BRTN (D), and all 4 breast cancer types (E) based on their microbial signature detection pattern. The endocrine receptor (estrogen/progesterone) positive breast cancers are abbreviated as BRER, human epidermal growth factor receptor 2 positive breast cancers are abbreviated as BRHR, triple positive (estrogen, progesterone, and HER2 receptor positive) breast cancers are abbreviated as BRTP and the triple negative (absence of estrogen, progesterone, and HER2 receptors) breast cancers are abbreviated as BRTN.

Figure 5

Heat map of hierarchical clustering of the 4 types of breast cancers (A), and the proportion of patients with and without severe outcome (death) in each cluster/sub-cluster (B). Among the breast cancer samples, the endocrine receptor (estrogen/progesterone) positives are abbreviated as BRER, human epidermal growth factor receptor 2 positives are abbreviated as BRHR, triple positives (estrogen, progesterone, and HER2 receptor positive) are abbreviated as BRTP and the triple negatives (absence of estrogen, progesterone, and HER2 receptors) are abbreviated as BRTN.

Figure 6

Box plots representing either significant or trend of higher detection of microbial signatures in BRTN (A), BRTP (B), BRER (C), and BRHR (D) cases with low (alive) or severe (dead) clinical outcomes, compared by one sided t-test. The p-value of the tests is shown in the figure if significant. NS represents non-significant test, however still a trend cannot be ignored. Among the breast cancer types, the endocrine receptor (estrogen/progesterone) positives are abbreviated as BRER, human epidermal growth factor receptor 2 positives are abbreviated as BRHR, triple positives (estrogen, progesterone, and HER2 receptor positive) are abbreviated as BRTP and the triple negatives (absence of estrogen, progesterone, and HER2 receptors) are abbreviated as BRTN.

Figure 7

PCR validation of microbial signatures in the 4 types of breast cancers and healthy control, using the primers from Table 7. Among the breast cancer types, the endocrine receptor (estrogen/progesterone) positives are abbreviated as BRER, human epidermal growth factor receptor 2 positives are abbreviated as BRHR, triple positives (estrogen, progesterone, and HER2 receptor positive) are abbreviated as BRTP and the triple negatives (absence of estrogen, progesterone, and HER2 receptors) are abbreviated as BRTN. The breast control samples obtained from healthy individuals are abbreviated as NC. The left shows the cropped gel pictures of EtBr stained amplicons run on agarose gel, where M is DNA ladder of RsaI digested ϕX/174, NTC is non-template control. The sequenced amplicons were subjected to nucleotide blast program in NCBI, and the results are shown in the right. In the Polyomavirus PCR gel picture, the orange and the green arrow heads signify Simian virus 40 and Merkel cell polyomavirus amplicons respectively, the electropherogram of the sequences of which are marked with the same arrow heads in Supplementary Figure S3.