The Predictive Utility of MammaPrint and BluePrint in Identifying Patients with Locally Advanced Breast Cancer Who are Most Likely to Have Nodal Downstaging and a Pathologic Complete Response After Neoadjuvant Chemotherapy

Background Neoadjuvant chemotherapy (NCT) increases the feasibility of surgical resection by downstaging large primary breast tumors and nodal involvement, which may result in surgical de-escalation and improved outcomes. This subanalysis from the Multi-Institutional Neo-adjuvant Therapy MammaPrint Project I (MINT) trial evaluated the association between MammaPrint and BluePrint with nodal downstaging. Patients and Methods The prospective MINT trial (NCT01501487) enrolled 387 patients between 2011 and 2016 aged ≥ 18 years with invasive breast cancer (T2–T4). This subanalysis includes 146 patients with stage II–III, lymph node positive, who received NCT. MammaPrint stratifies tumors as having a Low Risk or High Risk of distant metastasis. Together with MammaPrint, BluePrint genomically (g) categorizes tumors as gLuminal A, gLuminal B, gHER2, or gBasal. Results Overall, 45.2% (n = 66/146) of patients had complete nodal downstaging, of whom 60.6% (n = 40/66) achieved a pathologic complete response. MammaPrint and combined MammaPrint and BluePrint were significantly associated with nodal downstaging (p = 0.007 and p < 0.001, respectively). A greater proportion of patients with MammaPrint High Risk tumors had nodal downstaging compared with Low Risk (p = 0.007). When classified with MammaPrint and BluePrint, more patients with gLuminal B, gHER2, and gBasal tumors had nodal downstaging compared with HR+HER2−, gLuminal A tumors (p = 0.538, p < 0.001, and p = 0.013, respectively). Conclusions Patients with genomically High Risk tumors, defined by MammaPrint with or without BluePrint, respond better to NCT and have a higher likelihood of nodal downstaging compared with patients with gLuminal A tumors. These genomic signatures can be used to select node-positive patients who are more likely to have nodal downstaging and avoid invasive surgical procedures. Supplementary Information The online version contains supplementary material available at 10.1245/s10434-023-14027-9.

achieved a pathologic complete response.MammaPrint and combined MammaPrint and BluePrint were significantly associated with nodal downstaging (p = 0.007 and p < 0.001, respectively).A greater proportion of patients with MammaPrint High Risk tumors had nodal downstaging compared with Low Risk (p = 0.007).When classified with MammaPrint and BluePrint, more patients with gLuminal B, gHER2, and gBasal tumors had nodal downstaging compared with HR+HER2−, gLuminal A tumors (p = 0.538, p < 0.001, and p = 0.013, respectively).Conclusions.Patients with genomically High Risk tumors, defined by MammaPrint with or without BluePrint, respond better to NCT and have a higher likelihood of nodal downstaging compared with patients with gLuminal A tumors.These genomic signatures can be used to select node-positive patients who are more likely to have nodal downstaging and avoid invasive surgical procedures.nodal involvement, which may increase the likelihood of breast-conserving surgery and targeted axillary dissection (TAD), reducing surgical morbidity and improving longterm outcomes. 2,38][9] Axillary lymph node dissection (ALND) provides accurate nodal staging information but has increased associated morbidities, most notably, lymphedema. 10,111][12] However, SLNB can have high false negative rates, leaving the management of clinically node-positive patients debatable. 10,13More recently, targeted axillary dissection (TAD), which incorporates SLNB and radio-or carbon-tracing to selectively remove positive nodes, has been increasingly utilized by some surgeons to improve the accuracy of axillary staging without compromising clinical outcome in clinically node-positive patients. 14,15ere, we considered whether genomic information from the breast primary tumors could be used to identify patients who are likely to have nodal downstaging with NCT and thus be good candidates for less invasive nodal staging procedures, such as TAD.MammaPrint, a 70-gene signature (70-GS) that classifies patients as having a High Risk or Low Risk of distant metastasis, provides predictive and prognostic information for patients with early stage breast cancer and can identify Low Risk patients who may safely avoid chemotherapy. 16,17BluePrint is an 80-gene molecular subtyping signature that genomically (g) categorizes tumors as Luminal-Type (gLuminal), human epidermal growth factor 2 (HER2)-Type (gHER2), or Basal-Type (gBasal) on the basis of underlying signaling pathways. 18Together, MammaPrint and BluePrint identify molecular subtypes that more precisely determine therapeutic benefit in patients with early stage breast cancer than clinical factors alone, and this genomic classification corresponds better with 5-year outcomes and neoadjuvant chemosensitivity. 19,20he Multi-Institutional Neo-adjuvant Therapy MammaPrint Project I (MINT) trial was designed to evaluate the ability of MammaPrint and BluePrint to predict responsiveness to NCT in patients with locally advanced breast cancer.In this post hoc subanalysis from the MINT trial, we assessed the ability of MammaPrint and BluePrint to determine the likelihood of nodal downstaging after NCT.

Patient Cohort
Patients (N = 387) aged ≥ 18 years with histologically confirmed invasive breast cancer were prospectively enrolled into MINT (NCT01501487) between 2011 and 2016 across 17 US institutions.Among the enrolled patients with T2-T4 invasive breast cancer, 321 had MammaPrint and BluePrint testing performed (Fig. 1).The current study is a post hoc subanalysis to evaluate nodal downstaging

Clinical and Molecular Subtyping
Preoperative core biopsies were used to assess hormone receptor (HR) and HER2 status by immunohistochemistry (IHC) or IHC/fluorescence in situ hybridization (FISH), respectively, per American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. 21,22IHC/FISH were performed under central pathology review at the University of South Florida.Clinical risk was determined using Adjuvant!Online (AOL) using MINDACT guidelines. 16The same core biopsies were used to isolate RNA for MammaPrint and BluePrint signatures, which were performed at Agendia (Irvine, CA, USA).Tumors were classified with MammaPrint as Low Risk (MammaPrint index > 0.000) or High Risk (MammaPrint index ≤ 0.000). 23BluePrint categorizes tumors by subtype (gLuminal, gHER2, or gBasal). 18

Objectives and Endpoints
The primary objective of the trial was to determine chemosensitivity prediction using MammaPrint and BluePrint.In MINT, therapeutic response was evaluated by pCR, defined as the absence of invasive carcinoma in the breast and axilla by microscopic examination of the resected specimen, regardless of the presence of carcinoma in situ (ypT0/Tis, ypN0).The objective of this post hoc subanalysis was to assess the ability of MammaPrint and BluePrint to predict the likelihood of nodal downstaging as well.

Statistical Analysis
Patient clinical characteristics were summarized using descriptive statistics.Differences in age were evaluated by one-way analysis of variance (ANOVA), and other clinical characteristics were assessed by either chi-squared or Fisher's exact test.The association of pCR or nodal downstaging with MammaPrint risk, combined MammaPrint and BluePrint subtype, or other clinical factors were assessed using a two-tailed proportional z-test.Statistical significance was defined by two-sided p < 0.05 for all tests.All statistical analyses were conducted using GraphPad Prism (version 9.0.2) and R (version 4.1.1).

DISCUSSION
Achieving a pCR has been associated with better outcomes and is considered by many to be a surrogate for long-term survival. 6,7,24Nodal downstaging, which can be achieved with NCT, has also been associated with better outcomes. 7pCR and nodal downstaging following NCT improves the rates of breast-conserving therapy and may de-escalate invasive surgical procedures.Accordingly, it is important to identify patients who may be candidates for receiving NCT and will likely achieve axillary downstaging to directly reduce surgery-associated morbidities.This is the first study using the genomic classifiers MammaPrint and BluePrint to identify patients who may be good candidates for less invasive surgical procedures such as TAD.
In this study, the rates of complete nodal downstaging (ypN0) were comparable to pCR rates for each combined MammaPrint and BluePrint subtype.No patients with gLuminal A tumors had a pCR in the breast and nodes, which is in line with results from recent NBRST and NBREaST-II studies. 19,25Although there are only a small number of patients with gLuminal A tumors included in this study, they exhibited the lowest rate of complete nodal downstaging with NCT in comparison with MammaPrint and BluePrint High Risk tumors.We observed variable responses in patients with gLuminal B tumors, with 11.1% achieving a pCR and 55.6% (n = 25/45) having a partial response in the primary tumor with NCT.Additionally, 26.7% of patients with gLuminal B tumors had nodal downstaging with NCT.
FIG. 3 Association of nodal downstaging by MammaPrint and BluePrint; (a) MammaPrint Index was plotted for tumors from patients with nodal downstaging (cN+ to ypN0) and compared with residual disease tumors (cN+ to ypN+), (b) proportion of patients with Low Risk tumors having nodal downstaging (ypN0) and residual disease (RD) was compared with those with High Risk tumors, (c) patients were also categorized by BluePrint molecular subtype and MammaPrint Index was plotted for patients with ypN0 or RD for each molecular subtype, for gLuminal A tumors, MammaPrint indices are only shown for HR+HER2− tumors, (d) percentage of patients who achieved nodal downstaging for each BluePrint subtype, proportion of patients with gLuminal B, gHER2, and gBasal tumors who achieved ypN0 were compared with patients with gLuminal A tumors (the frequency of nodal downstaging for gLuminal A is shown for patients with HR+HER2− tumors), (e) within gHER2 and gBasal tumors, the percent of patients with nodal downstaging and residual disease are shown by hormone status, (f) percentage of patients who achieved pCR for each BluePrint subtype, proportion of patients with gLuminal B, gHER2, and gBasal tumors who achieved a pCR were compared with patients with gLuminal A tumors, significance between continuous variables (MammaPrint Index) was assessed using unpaired t-test or one-way ANOVA, two-tailed proportional z-test was used to compare categorical subgroups and between hormone status ◂ Together, these data suggest that there is greater genomic diversity among patients with Luminal tumors, which can be distinguished with MammaPrint.Patients with gLuminal A and gLuminal B tumors both have underlying luminal biology, but gLuminal B tumors are considered High Risk with MammaPrint.Therefore, it is consistent with the characteristics of gLuminal B tumors to demonstrate lower response rates compared with other MammaPrint HighRisk tumor subtypes (gHER2 and gBasal) but to have higher response rates compared with gLuminal A tumors.These data also suggest that more effective preoperative therapies than endocrine therapy and standard chemotherapy are needed for patients with gLuminal B tumors.Thus, including these patients in trials evaluating new targeted therapies, such as CDK4/6 inhibitors or HER2 antibodydrug conjugates, should be considered.In contrast to patients with gLuminal A tumors, patients with MammaPrint High Risk or combined MammaPrint and BluePrint HER2-Type and Basal-Type tumors had significantly superior responses in the nodes and primary tumor.The majority of patients with gHER2 and gBasal tumors achieved complete nodal downstaging, of whom 78.6% (n = 22/28) and 59.1% (n = 13/22) also had a breast pCR, respectively.These results are consistent with previous studies in which more patients with clinically defined HER2+ and TN breast cancer achieve a pCR and nodal downstaging than with HR+HER2− or clinically-defined luminal tumors. 5,7,26,27Interestingly, the proportion of patients who achieved nodal downstaging within gHER2 and gBasal tumors were independent of HR status, which is in line with our recent report of patients with HR+/Basal tumors having distinct pCR rates and outcomes compared with patients with HR+/Luminal tumors. 20Importantly, there was a 15.8% discordance between clinical risk and MammaPrint risk assessment, where these clinically high-risk patients were identified as MammaPrint Low Risk.While MammaPrint is generally indicated for patients with cN1 disease, a higher proportion of patients with MammaPrint High Risk cN2-cN3 tumors achieved nodal downstaging (complete and partial) in comparison with MammaPrint Low Risk cN2-cN3 tumors, which is consistent with the cohort overall (including cN1).In patients with gLuminal A tumors, chemotherapy would still be indicated if surgical pathology was N2 or greater.Together, these data emphasize the expanded utility of using MammaPrint and BluePrint genomic testing, which can further characterize traditional clinical subtyping.
One limitation of this study is that we do not have long-term outcome data yet to correlate with BluePrint and MammaPrint results.If pCR is an accepted surrogate for long-term outcomes, we would expect that patients, particularly those with genomically HER2-Type and Basal-Type tumors who achieve pCR, will have a better prognosis compared with those with residual disease.This study is also limited by the small number of patients eligible for inclusion, and therefore a larger cohort would be recommended to confirm the results from this study.

CONCLUSIONS
This is the first study to show that nodal downstaging can be predicted by MammaPrint and BluePrint beyond achieving a pCR or clinical factors such as HR status.Genomically High Risk patients, identified by MammaPrint with or without BluePrint molecular subtyping, had higher proportions of nodal downstaging than in the overall lymph node-positive cohort.Our data suggest that treating patients with clinically HR+HER2−, genomically Luminal A-Type tumors with chemotherapy for the purpose of nodal downstaging is likely to yield suboptimal results, and these patients should be counseled on this prior to treatment, whereas those with MammaPrint High Risk tumors respond better to NCT.Overall, results from this study suggest that MammaPrint and BluePrint may help identify good candidates for less invasive axillary procedures such as TAD, which may reduce surgery-associated complications and radiation and improve patient outcomes.
The Predictive Utility of MammaPrint … 0.790), preoperative clinical tumor size (p = 0.526), and clinical lymph node status (p = 0.434) were not associated with nodal downstaging (Table

TABLE 2
pCR pathological complete response, cN clinical nodal stage, ypN pathological nodal stage after neoadjuvant chemotherapy