Developing MenaCalc: an assay to predict risk of breast cancer tumor metastasis through quantification of Mena protein isoforms
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Metastasis is the leading cause of poor prognosis for individuals diagnosed with cancer. Breast cancer is particularly prevalent with 1 in 10 women receiving a breast cancer diagnosis in her lifetime. There are various types of breast cancers that are distinguished by molecular subtype as defined by specific biomarker expression profiles: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). The subtypes defined by the varying expression of these receptors respond differently to cancer treatments. For example, luminal A ([ER/PR+] HER2- KI67-) responds well to endocrine therapy and patients generally have a good prognosis, whereas triple negative breast cancer (TNBC) ([ER/PR-] HER2- basal marker+) has no specific targeted treatment available and the prognosis is usually poor. Patients with the HER2 subtype often develop resistance to the treatment specific to the breast cancer molecular subtype. Since 90% of all cancer-related deaths are due to metastatic disease, effectively treating all of these types of breast cancers before metastasis is an important factor in achieving a more positive outcome, In order for metastasis to occur, a tumor cell must have the ability to mobilize, intravasate into the vasculature, and then extravasate and proliferate into a tumor at a distant site. Numerous biological and environmental factors must facilitate each of these steps in order for metastasis to occur. One biomarker of metastasis is a tumor microenvironment of metastasis (TMEM). A TMEM is the physical apposition of a Mena-expressing tumor cell, a macrophage (a type of white blood cell), and an endothelial cell (a blood vessel cell). Each TMEM component plays a key role in breast cancer biology. The automated clinical assay MetaSite BreastTM was developed by MetaStat, Inc. to quantify TMEMs. The MetaSiteTM score directly correlates with risk of developing metastasis. The Mena protein is involved in cell motility and expressed isoforms can either promote metastasis (for example, MenaINV), or protect and prevent metastasis (for example, Mena11a). These isoforms are not expressed in a binary manner and studies have shown that the ratio of MenaINV to Mena11a can give insight into the pro-metastatic/anti-metastatic biology of the cell. To indirectly measure the amount of MenaINV, the Z-score of Mena11a is subtracted from the Z-score of pan-Mena (all Mena isoforms), yielding a theoretical maximum amount of MenaINV, called Menacalc. This process is performed by quantitative analysis of multiplexed immunofluorescence staining through the MenaCalcTM assay developed by MetaStat, Inc. The results of this study demonstrated that MenaCalcTM is a high-performing, high-throughput assay that was clinically validated under CLIA-approved protocol in January 2016. The assay surpassed all benchmark goals for precision and performance. For both day-to-day and run-to-run operations, precision and reproducibility were analyzed using Pearson’s R and slope. The day-to-day reproducibility yielded Pearson’s R values of 0.879 and 0.853 comparing Day 1 vs. Day 2 and Day 2 vs. Day 3, respectively. The slopes for the same comparisons were 0.985 and 0.982, respectively. The analysis of run-to-run precision had Pearson’s R values of 0.999 and 0.994 comparing Day 1 vs. Day 2 and Day 2 vs. Day 3, respectively. The slopes were 0.999 for both comparisons. The development of such an assay brings new elements of precision and reproducibility to the current market of breast cancer biomarker tests. Statistical analysis revealed a wide range of MenaCalcTM scores that were independent of total Mena expression. Individual images showed a range of MenaCalcTM values from a low of only 2.9% of cells with a high MenaCalcTM score to a high of 97.4% of cells with a high MenaCalcTM score. Regions of high MenaCalcTM scores correlated with areas of invasive tumor. Preliminary data assessing the synergistic use of both the MetaSite BreastTM and the MenaCalcTM assays were promising. These data suggests that both physical MetaSiteTM structures and protein expression levels can be used to more thoroughly understand the biology of breast cancer and the path to metastasis. Three clusters of combined MetaSiteTM/MenaCalcTM scores were observed: MetaSiteTM low/MenaCalcTM low, MetaSiteTM low/MenaCalcTM high, MetaSiteTM high/MenaCalcTM high. Because a MetaSiteTM High/MenaCalcTM Low score combination was not observed, a high MenaCalcTM score may be necessary for TMEM formation. Studies are ongoing to further evaluate the synergy of the MetaSite BreastTM and the MenaCalcTM in order to bring more power to the assessment of metastatic risk.