Options for Breast Cancer Prevention in High-Risk Patients

Breast cancer is the most frequently diagnosed non-skin cancer in women, and one in eight women will develop breast cancer within their Iifetimes. Unfortunately, the strongest risk factors for breast cancer (i.e. age, family history, hormonal factors) are not easily modified. There is some evidence that chemopreventative drugs may be able to prevent breast cancer in high-risk patients. Tamoxifen and raloxifene have been shown to reduce the risk of breast cancer in high-risk women but may be associated with several serious adverse events. Clinical trials are currently in progress to determine if aromatase inhibitors are a viable alternative for breast cancer prevention, as they may be considered effective in the early treatment of breast cancer. For patients with BRCA1 and BRCA2 mutations, a bilateral prophylactic mastectomy may be an option. This article discusses the risks and benefits of available treatment options for breast cancer prevention in high-risk patients

Number of mutations per sample.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.</div

Frequency of mutations detected in samples passaged in different cell lines and with and without antiserum selection pressure (see Antiserum Pressure section Table 3).

Frequency of mutations detected in samples passaged in different cell lines and with and without antiserum selection pressure (see Antiserum Pressure section Table 3).</p

Neutralization assay, GFP expression results.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.</div

Sequence read coverage depth for sequences derived from neutralization assay.

Sequence read coverage depth for sequences derived from neutralization assay.</p

Comparison of variant frequencies associated with cell culture passage, passage under antiserum selection, and mAb selection.

Mutations that are present in at least 1 sample at > 5% are shown and that are present in antiserum passaged samples and/or neutralization data are shown (see S1 Table for mutations present at 1% or greater). Note cell passaged samples from Vero and Caco-2 cells did not have mutations that persisted and are not shown. Antiserum passaged samples (VA-P6) have serum dilution (1:5 or 1:10) shown in parentheses. Samples that were tested with the neutralization assay are denoted by the mAb name, the viral inoculum (UP, VO-P6, or VA-P6), and the mAb concentration (5 μg/ml, 10 μg/ml). Data from virus only passaged samples (no antibody selection) are shown in blue cells and data from virus passaged under antiserum or mAb selection are in yellow shaded cells. Top row cells shaded grey signify columns with data from both virus only and antibody selection. With the exception of Vero P4, Vero-T P4 and Caco P4, all samples were processed (PCR and sequencing) in duplicate; the percent frequency is an average of the two values.</p

Mutation distribution among samples passaged with or without antibody selection pressure.

Mutation distribution among samples passaged with or without antibody selection pressure.</p

Mutations detected after passage in cell lines.

Abbreviations Vero-T: Vero/TMPRSS2; NT POS: Nucleotide position in the reference sequence Wuhan-Hu-1 (NC_045512.2); AVG FREQ: Average frequency; TOTAL DP: Depth of sequence read coverage for the nt pos; REF AA: Amino acid in the reference sequence; ALT AA: Amino acid in the sample sequence; AA POS: Residue number in the spike gene.</p

Virus passage and assay workflow.

Cell lines and assay components are in boxes, viral inocula are in ovals. UP: unpassaged virus; SEQ: samples were deep sequenced; VERO-TMP: VERO/TMPRSS2 cell line; P4 mix: mixture of virus from passage 4 all cell lines (Vero, Vero/TMPRSS2, and Caco-2); VO: virus only (no antibody selection), VA: virus passaged in the presence of antibody (antiserum from vaccinated individuals).</p