Where did we start from

Tumor recurrences are one of the biggest risk factors for breast cancer patients. Determining which tumors are likely to recur and what way they will likely evolve is crucial not only to understand cancer biology, but to tailor therapies to patients. Ipsilateral breast tumor recurrences (IBTRs; defined as tumors recurring in the same breast) are an important event that constitute a therapeutic window to prevent further cancer spread.

While the genomic characteristics and their effect on transcript and protein expression has been well characterized in primary tumors (PTs) and distant metastases, IBTRs remain understudied.  With this in mind, we have employed a previously developed proteogenomics workflow to determine the evolution of IBTRs at the genomic, transcriptomic, and proteomic level from corresponding matched PTs to investigate paths of mutational evolution. On top of this, we compared PTs that returned as an IBTR with primaries that did not recur so to detect tumor characteristics predisposing of IBTR.

The evolution of ipsilateral breast tumor recurrences impacts genes and proteins differently

We analyzed the change in genomic characteristics between IBTR and PT pairs, investigating changes in mutational processes, gene copy number and cancer gene variants. Here we found a relationship between the lack of expression of the estrogen receptor (ER) and an increase in the aforementioned genomic features. When looking downstream of the genome, more specifically into the transcriptome (RNA) and proteome (proteins), we noticed a much more pronounced dissimilarity between IBTRs and their respective PTs, suggesting that changes in the genome promote even bigger variation at the RNA and protein levels. On top of this, lack of ER and highly proliferation levels related to a much higher dissimilarity, hence a more pronounced evolution, between IBTRs and their originating PTs.

Ipsilateral breast tumor recurrences evolve in connection with hormonal status

Further down this road, comparison between ER positive and negative tumors showed that ER negative IBTRs were enriched in genes and proteins involved in cell cycle, DNA replication, and RNA transcription over their ER positive counterparts. Among the genes highly associated with proliferation and the accumulation of mutational features, the APOBEC family member APOBEC3B was found highly enriched in ER negative tumors and well correlated with the tumor proliferation marker Ki-67. From this we hypothesize that APOBEC is not only a determinant of the mutational burden in (breast) cancer, but also an important co-factor in the evolution of tumor cells over the course of the disease.

Proliferation rate and immune involvement are associated to likelihood to ipsilateral breast tumor recurrence

In order to understand the mechanisms underlying IBTR formation we compared the transcriptome and the proteome of PTs that developed an IBTR with a set of primaries that did not. By employing pathway analysis we detected an enrichment of proliferation and immune signaling pathways in PTs that experienced a recurrence. These results were well recapitulated by immunohistochemical analysis of the tumor proliferation marker Ki-67 and tissue leukocyte counts, with good correlation and the prospect of being combined into a prognostic score.

Take home and outlook

Understanding the mechanisms of tumor evolution and they key elements behind it are paramount in defining therapeutic intervention. As our data pointed at APOBEC3B as a major determinant of genetic changes in IBTRs, we believe it would be worthwhile investigating its role in IBTR evolution and its relationship with clinical outcomes.

Concomitantly, determining the factors that lead to IBTR formation would be important in patient screening practice, thus evaluation of immune cell infiltration and proliferation rates offer a good starting point to define new prognostic markers.

Manuscript DOI: 10.1038/s42003-023-04526-6