Functional buffering that ensures biological robustness is critical for maintaining tissue homeostasis, organismal survival, and evolution of novelty. However, the mechanism underlying functional buffering, particularly in multicellular organisms, remains largely elusive. Here, we proposed that functional buffering can be mediated via expression of buffering genes in specific cells and tissues, by which we named Cell-specific Expression-BUffering (CEBU). We developed an inference index (C-score) for CEBU by computing C-scores across 684 human cell lines using genome-wide CRISPR screens and transcriptomic RNA-seq. We report that C-score-identified putative buffering gene pairs are enriched for members of the same duplicated gene family, pathway, and protein complex. Furthermore, CEBU is especially prevalent in tissues of low regenerative capacity (e.g., bone and neuronal tissues) and is weakest in highly regenerative blood cells, linking functional buffering to tissue regeneration. Clinically, the buffering capacity enabled by CEBU can help predict patient survival for multiple cancers. Our results suggest CEBU as a potential buffering mechanism contributing to tissue homeostasis and cancer robustness in humans.

Background: The DESTINY-Breast04 clinical trial demonstrated the superiority of trastuzumab deruxtecan (T-DXd) versus physician’s choice chemotherapy in HER2-low [immunohistochemistry (IHC) staining score 1+ or 2+ with no gene amplification by in-situ hybridization] metastatic breast cancer. The reproducibility of HER2 IHC category 0 versus 1+ scoring is poor in community practice. 89% of the DESTINY-04 participants were hormone receptor positive (HR+). In HR+ breast cancers, the Oncotype DX® (ODX) assay is widely used to estimate risk of recurrence and guide adjuvant chemotherapy selection. It also provides standardized quantification of HER2 mRNA expression by RT-PCR. Prior studies have demonstrated a high degree of overall concordance between central IHC and RT-PCR using ODX in HER2+ and HER2- cases. Here, we (i) compare HER2 mRNA levels quantified by ODX in HER2 IHC 0, 1+ and 2+ invasive breast carcinomas (IBC), (ii) compare the Recurrence Score® (RS) distribution across these three IHC categories and (iii) describe RS distribution and proliferation score in HER2-low IBC. Methods: 212 patients with HER2 IHC 0, 1+ and 2+ who were negative for HER2 gene amplification by FISH and had RS results were identified in Yale Department of Pathology archives. All US samples submitted for IBC ODX testing between 2005 to 2021 were reviewed in the Exact Sciences database. RS, quantitative HER2 mRNA expression, and proliferation scores were examined. IHC results were not available. Based on quantitative RT-PCR measures of HER2 expression, cases were assigned to: HER2 positive ≥11.5, equivocal ≥10.7 to < 11.5, and negative < 10.7 (1 unit increment is equivalent to approximately 2-fold change). Results: In the Yale cohort, 42%, 33%, and 25% of cases were IHC 0, 1+ and 2+, respectively. There was no difference in age or tumor grade by IHC category. HER2 mRNA levels increased across IHC categories (means 9.05, 9.16, 9.39, respectively), but in group-wise comparisons, only the IHC 0 compared to IHC 2+ reached statistical significance (Mann-Whitney test, p=0.0014). The RS scores were also modestly, but significantly higher in IHC 2+ compared to IHC 0 cases (mean 19.5 vs 14.51, Mann-Whitney p=0.034). Among IHC 0 and 1+ cases, 14% had RS >25, and among IHC 2+ cases, 32% had RS >25. All IHC 0 and 1+ cases were HER2 negative by RT-PCR. Of the HER2 2+ cases, there was one HER2 positive and one HER2 equivocal by RT-PCR. There was substantial variation in HER2 expression by RT-PCR in all IHC groups. In the Exact Sciences cohort, a total of 957,624 samples were analyzed. 0.8% of samples were HER2 positive, 1.2% were equivocal, and 98% were negative by RT-PCR. There was a wide range of RS results. Of the HER2 positive cases, 94.7% had RS >25. Among the HER2 equivocal cases 39.1% had RS >25, and among the HER2 negative cases, 15.5% had RS >25. Conclusions: HER2 IHC 0 and HER2 low IBC have a broad and overlapping range of HER2 expression by RT-PCR. Whether RT-PCR based HER2 expression predicts benefit from T-DXd is yet to be determined. Most HER2 low cases have RS ≤25, indicating no or limited benefit from adjuvant chemotherapy. Further studies are required to determine if patients with HER2 low IBC and RS ≤25, particularly with high anatomical risk, could benefit from adjuvant T-DXd. Table 1. Clinicopathologic and molecular characteristics by HER2 IHC group Table 2. Quantitative gene expression by HER2 IHC group Citation Format: Mariya Rozenblit, Hao-Kuen Lin, Nhu Thuy Can, Cynthia A. Flannery, Jess Hoag, Alekhya Akkunuri, Helen Bailey, Frederick Baehner, Lajos Pusztai. Molecular characterization of HER2-low invasive breast carcinoma by quantitative RT-PCR using Oncotype DX® [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-14.

Functional buffering ensures biological robustness critical for cell survival and physiological homeostasis in response to environmental challenges. However, in multicellular organisms, the mechanism underlying cell- and tissue-specific buffering and its implications for cancer development remain elusive. Here, we propose a Cell-specific Expression-BUffering (CEBU) mechanism, whereby a gene's function is buffered by cell-specific expression of a buffering gene, to describe functional buffering in humans. The likelihood of CEBU between gene pairs is quantified using a C-score index. By computing C-scores using genome-wide CRISPR screens and transcriptomic RNA-seq of 684 human cell lines, we report that C-score-identified putative buffering gene pairs are enriched for members of the same pathway, protein complex and duplicated gene family. Furthermore, these buffering gene pairs contribute to cell-specific genetic interactions and are indicative of tissue-specific robustness. C-score derived buffering capacities can help predict patient survival in multiple cancers. Our results reveal CEBU as a critical mechanism of functional buffering contributing to cell survival and cancer robustness in humans.