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Objectives We assessed the interobserver and interantibody reproducibility of HER2 immunohistochemical scoring in an enriched HER2-low–expressing breast cancer cohort. Methods A total of 114 breast cancer specimens were stained by HercepTest (Agilent Dako) and PATHWAY anti-HER2 (4B5) (Ventana) antibody assays and scored by 6 breast pathologists independently using current HER2 guidelines. Level of agreement was evaluated by Cohen κ analysis. Results Although the interobserver agreement rate for both antibodies achieved substantial agreement, the average rate of agreement for HercepTest was significantly higher than that for the 4B5 clone (74.3% vs 65.1%; P = .002). The overall interantibody agreement rate between the 2 antibodies was 57.8%. Complete interobserver concordance was achieved in 44.7% of cases by HercepTest and 45.6% of cases by 4B5. Absolute agreement rates increased from HER2 0-1+ cases (78.1% by HercepTest and 72.2% by 4B5; moderate agreement) to 2-3+ cases (91.9% by HercepTest and 86.3% by 4B5; almost perfect agreement). Conclusions Our results demonstrated notable interobserver and interantibody variation on evaluating HER2 immunohistochemistry, especially in cases with scores of 0-1+, although the performance was much more improved among breast-specialized pathologists with the awareness of HER2-low concept. More accurate and reproducible methods are needed for selecting patients who may benefit from the newly approved HER2-targeting agent on HER2-low breast cancers.

In light of the significant clinical benefits of novel HER2-targeting antibody-drug conjugates in advanced HER2-low expressing breast cancers in recent phases I and III clinical trials, particularly trastuzumab-deruxtecan (T-Dxd), the new "HER2-low" category in breast cancers (breast cancer with a HER2 IHC score of 1+, or 2+ without gene amplification) has gained increasing attention. In the past year, "HER2-low" breast cancers have been under active investigation by both oncologists and pathologists. In this current review, we update the recent cutting-edge research on HER2-low breast cancers, with a focus on the biology of HER2-low breast cancers, the issues on the identification of HER2-low breast cancers by immunohistochemistry in current practice of pathology, and the future directions in this emerging category in breast cancers.

Recently, clinical trials have demonstrated promising efficacy for novel HER2-targeted therapies in HER2-low breast cancers, raising the prospect of including a HER2-low category (immunohistochemical [IHC] score of 1+, or 2+ with non-amplified in-situ hybridization [ISH]) in the HER2 evaluation of breast cancers. In order to better understand this newly-proposed HER2 category, we investigated the incidence, HER2 staining patterns, clinicopathologic features, and genomic profile of HER2-low breast cancers. HER2-stained slides of 281 consecutive breast cancers were re-reviewed and the clinicopathologic information, MammaPrint, and BluePrint results of these cases were retrospectively analyzed. HER2-low breast cancers were identified in 31% of cases and were more common in estrogen receptor (ER)-positive than ER-negative breast cancers (33.6% vs 15%, p = 0.017). HER2-low cancers were generally clinical stages I-II (79%), ER-positive (93.1%), had homogenous HER2 staining (59.2%), HER2 IHC score of 1+ (87.4%), ductal phenotype (81.6%), histologic grades of 1 or 2 (94.2%) and luminal molecular subtypes (94.3%). Three HER2-low patients received neoadjuvant chemotherapy and none of them achieved pathologic complete response. When compared to HER2-negative (IHC of 0+) and HER2-positive (IHC of 3+ or IHC of 2+ with amplified ISH) cancers, HER2-low breast cancers had significantly lower Ki-67 (p = 0.03 and p 0.01, respectively) and higher ER positivity (p = 0.01 and p = 0.03, respectively). HER2-low breast cancers were less likely to be basal molecular subtype when compared to HER2-negative cancers (p 0.01) and were less likely to have a HER2 molecular subtype when compared to HER2-positive cancers (p 0.01). When adjusted for ER status, there was no significant difference on all the examined variables between HER2-low and HER2-negative groups. Our study provides valuable baseline characteristics of HER2-low breast cancers deriving from consecutive, real-world cases with a consensus confirmation of HER2 status, and would help to increase our understanding of this newly-proposed HER2 category in breast cancers.

Introduction: Multigene genomic profiling has become the standard of care in the clinical risk-assessment and risk-stratification of ER+, HER2− breast cancer (BC) patients, with Oncotype DX® (ODX) emerging as the genomic profile test with the most support from the international community. The current state of the health care economy demands that cost-efficiency and access to testing must be considered when evaluating the clinical utility of multigene profile tests such as ODX. Several studies have suggested that certain lower risk patients can be identified more cost-efficiently than simply reflexing all ER+, HER2− BC patients to ODX testing. The Magee equationsTM use standard histopathologic data in a set of multivariable models to estimate the ODX recurrence score. Our group published the first outcome data in 2019 on the Magee equationsTM, using a modification of the Magee equationsTM combined with an algorithmic approach—the Rochester Modified Magee algorithm (RoMMa). There has since been limited published outcome data on the Magee equationsTM. We present additional outcome data, with considerations of the TAILORx risk-stratification recommendations. Methods: 355 patients with an ODX recurrence score, and at least five years of follow-up or a BC recurrence were included in the study. All patients received either Tamoxifen or an aromatase inhibitor. None of the patients received adjuvant systemic chemotherapy. Results: There was no significant difference in the risk of recurrence in similar risk categories (very low risk, low risk, and high risk) between the average Modified Magee score and ODX recurrence score with the chi-square test of independence (p > 0.05) or log-rank test (p > 0.05). Using the RoMMa, we estimate that at least 17% of individuals can safely avoid ODX testing. Conclusion: Our study further reinforces that BC patients can be confidently stratified into lower and higher-risk recurrence groups using the Magee equationsTM. The RoMMa can be helpful in the initial clinical risk-assessment and risk-stratification of BC patients, providing increased opportunities for cost savings in the health care system, and for clinical risk-assessment and risk-stratification in less-developed geographies where multigene testing might not be available.

Apoplectic leiomyomas—benign uterine leiomyomas with morphologic changes including hemorrhage, hypercellularity, mitotic activity, nuclear atypia, and even necrosis—can be difficult to distinguish from uterine leiomyosarcomas. Apoplectic leiomyomas have been associated with hormonal therapy; however, the relationship between apoplectic leiomyomas, hormones, and ethnicity has not received much attention in the literature. We evaluated the relationship of hormonal therapy and ethnicity in 869 women with uterine leiomyomas, 136 of which qualified as apoplectic leiomyomas. Apoplectic leiomyomas were observed in 23.3% (49/210) of women exposed to hormonal therapy compared to 13.2% (87/659) of women not exposed to hormonal therapy (p 45 years of age (p

Objectives Recent clinical trials have demonstrated significant clinical benefits from novel therapeutic compounds in breast cancer patient with human epidermal growth factor receptor 2 (HER2) immunohistochemical (IHC) score of 1+ or 2+ and negative in situ hybridization (ISH) result. A new concept of “HER2-low” breast cancer has been proposed and applied in the recent and ongoing clinical trials. In this article, we review the literature on the topic of HER2-low breast cancer. Methods A literature search in PubMed was performed using key words related to HER2-low breast cancer. Major relevant studies that were presented in international breast cancer conferences were also included. Results HER2-low breast cancer is currently defined as breast cancer with HER2 IHC score of 1+ or 2+ and negative ISH result. It likely represents a group of tumors with significant biological heterogeneity. Reports of clinical activity using the next generation of HER2-targeting antibody-drug conjugates in HER2-low breast cancers suggest that some strategies of targeting HER2 might be effective in this patient population while raising considerable concerns over limitations in our current testing methodologies and our ability to accurately identify such patients. Conclusions The promising efficacy of novel HER2-targeted therapy in advanced HER2-low breast cancers has raised the possibility for changing the clinical interpretation of HER2 status in breast cancer to include a HER2-low category; however, the definition of HER2-low breast cancer, the corresponding reliable and accurate quantitative HER2 testing methodology, and the biology of HER2-low breast cancer remain poorly defined.

Background: MammaPrint (MP) is an FDA approved early breast cancer prognostic genomic assay that examines the expression levels of 70 genes and classifies clinically high-risk patients with early-stage breast cancer into low or high risk of distant metastasis. Current ASCO guidelines do not support using MP testing in clinically low-risk patients. Historically, assessing a patient’s clinical risk for breast cancer recurrence has relied on a subjective evaluation of histologic factors, immunohistochemical features, and clinical characteristics. Genomic assay testing provides more objective prognostic and predictive information than those more traditional methods; however, several studies have shown that statistical models using standard histologic variables can give valuable prognostic information, and might be used as an alternative approach to genomic assays for clinical risk-assessment and risk-stratification, particularly in lower risk breast cancer patients, potentially resulting in significant cost savings for health care systems. One of these statistical models, the average Modified Magee equation, has been shown to be prognostic for a low risk of recurrence in ER positive Her2 negative breast cancer patients who have an average Modified Magee score (aMMs) of ≤ 12. The aMMs has been shown to correlate well with the Oncotype DX recurrence score; however, there is limited data on the correlation between the aMMs and MP. In this study, we evaluated the concordance between the aMMs and the MP index (MPI). Methods: 222 consecutive patients with a new diagnosis of hormone receptor positive Her-2 negative breast cancer at the University of Rochester between April 2020 and December 2020 were sent for MP testing. The aMMs was determined for each patient based on the estrogen receptor status, progesterone receptor status, HER-2 status, Ki-67 proliferation index, Nottingham score, and tumor size. Based on the previous literature, patients were risk-stratified by the aMMs into three groups: 1) low risk (aMMs ≤ 12; n = 75); 2) low - intermediate risk (aMMs >12 and ≤ 18; n = 96); and, 3) high risk (aMMs >18, n = 51). Based on the previous literature, patients were risk-stratified by the MPI into two groups: 1) low risk (MPI 0 to +1); and, 2) high risk (MPI -1 to 0). The correlation between the aMMs and the MPI was evaluated using the Pearson correlation coefficient. Results: The aMMs showed a negative correlation (r= -0.55) with the MPI, consistent with a lower risk aMMs more likely being associated with a lower risk MPI. 92% of patients with an aMMs ≤ 12 (very low risk) were classified as low risk by MP. 72% of patients with an aMMs > 12 and ≤ 18 (low - intermediate risk) were classified as low risk by MP. 47% of patients with an aMMs > 18 (high risk) were classified as high risk by MP (Table 1). All patients with an aMMs ≥ 24.7 (n=8) were classified as high risk by MP. Conclusion: Our study suggests that hormone receptor positive, Her-2 negative breast cancer patients with a lower risk aMMs are likely to have a lower MPI, suggesting that these patients have a decreased risk for breast cancer recurrence. As the aMMs approaches higher risk, the concordance with the MPI decreases; however 100% of patients with an aMMs ≥ 24.7 were classified as high risk by MP. The aMMs can be helpful in clinical risk-assessment prior to making a decision about sending a patient specimen out for MP testing. Additional studies are warranted. Table 1.Risk of recurrence using the average Modified Magee score (aMMs) and the MammaPrint (MP) IndexaMMs Risk-stratification categoryMP Low risk n (%)MP High risk n (%)Very low (aMMs ≤ 12)69 (92%)6 (8%)Low - Intermediate (aMMs > 12 and ≤ 18)69 (72%)27 (28%)High (aMMs >18)27 (53%)24 (47%) Citation Format: Hani Katerji, Huina Zhang, David Hicks, Bradley M Turner. Clinical-risk assessment of ER positive, Her2 negative breast cancer patients: Correlation between the average modified magee score and mammaprint [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-06-03.

INTRODUCTION: In 2013, Klein and Dabbs et al. published three linear regression equations (the new Magee equations) using different combinations of standard histopathologic variables. In 2015, our group published a modification of the new Magee equations. Based on this modification, we published an algorithmic approach using an average Modified Magee equation. This algorithmic approach supported reflex Oncotype DX (ODX) testing based on several cutoff points using an average Modified Magee score. In 2019, we validated this algorithmic approach as the Rochester Modified Magee Algorithm (RoMMA) in a multi-institutional study, with outcome data in 247 patients, suggesting that ER positive breast cancer patients with an average Modified Magee score of ≤ 12 had a low risk of breast cancer recurrence. There has been limited published outcome data on the Magee equation since that 2019 study. We have further refined our risk-stratification approach, with additional outcome data in 416 ER positive breast cancer patients. METHODS: 416 patients with an ODX recurrence score who had at least five years of follow-up data or a breast cancer recurrence were included in the final outcome analysis (2008-2017). All patients received either Tamoxifen or an aromatase inhibitor. None of the patients received adjuvant systemic chemotherapy. The average Modified Magee score was calculated and patients were stratified into four risk-stratification categories: 1) very low, 2) low ≤ 50 years of age, 3) low > 50 years of age, and 4) high. We compared these four risk-stratification categories, with outcomes, between the average Modified Magee score and the ODX recurrence score. A p-value of < 0.05 was considered statistically significant. RESULTS: 27/416 (6.5%) patients had a recurrence of breast cancer. When comparing the same risk category groups, there was no significant difference between the average Modified Magee score and the ODX recurrence score (Table 1). CONCLUSION: Our study further reinforces that breast cancer patients can be confidently stratified into low and high risk recurrence groups using the average Modified Magee score. The average Modified Magee score may be an alternative to ODX for clinical risk-assessment and risk-stratification, particularly in lower risk patients, offering breast cancer patients increased access to clinical risk-assessment and risk-stratification, both domestically and internationally, with a potential significant cost savings for health care systems. A large prospective evaluation, similar to the studies done by ODX, using multi-institutional data or data from studies like the NSABP trial B-14 and the NSABP trial B-20, is necessary. Table 1.Risk-stratification categories and outcomes using the average Modified Magee score (aMMs) and the Oncotype DX recurrence score (ODXRS)RECURRENCENO RECURRENCEP-VALUEVERY LOW (N)aMMs ≤ 12 (76)1 (1.3)75 (98.7)0.65ODXRS < 11 (108)4 (3.7)104 (96.3)LOW ≤ 50 years of age (N)aMMs > 12, ≤ 18 (50)3 (6.0)47 (94.0)1.0ODXRS 11 - 15 (32)1 (3.1)31 (96.9)LOW >50 years of age (N)aMMs > 12, ≤ 18 (153)8 (5.2)145 (94.8)0.52ODXRS 11 - 25 (214)16 (7.5)198 (92.5)HIGH (N)aMMs > 18 (137)15 (10.9)122 (89.1)1.0ODXRS ≥ 16 - 25 (33)* and ODXRS > 25 (29)**6 (9.7)56 (90.3)* Patients ≤ 50 years of age with an ODXRS of ≥16 - 25. ** All patients with an ODXRS of > 25 Citation Format: Numbereye Numbere, Ioana Moisini, Ajay Dhakal, Kristin Skinner, Michelle Shayne, Mary Ann Gimenez Sanders, Huina Zhang, David Hicks, Bradley M Turner. Clinical risk-assessment, risk-stratification, and outcomes of ER positive, HER2 negative breast cancer patients using the Rochester modified Magee algorithm (RoMMA) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-06-07.

INTRODUCTION: There is dearth information on the long-term outcome and role of HER2 targeted therapy in breast cancer patients in the HER2 FISH group 3 category, which is defined as breast cancer with a HER2/CEP17 ratio < 2 and HER2 copy number ≥ 6 signals/cell. In this study, we report the clinicopathologic features and outcomes of breast cancer patients in the HER2 FISH group 3 category at our institution. METHODS: We identified 52/2,874 (1.8%) breast cancer patients with HER2 FISH group 3 results between 1/2007 and 3/2020. 28 of these 52 patients had available detailed clinicopathologic and follow-up data, with an average follow-up of 38.5 months. RESULTS: Most of the cases with group 3 FISH results were high grade ductal carcinomas with positive hormonal receptor expressions and equivocal HER2 expression by immunohistochemistry. Among all the clinicopathologic variables, only tumor size (p=0.048) significantly contributed to the poor clinical outcomes. HER2 copy number failed to show any significant association with histologic grade, tumor size, clinical stage, hormonal receptor status or disease outcomes. There was no statistically significant difference in disease outcome between patients who were treated with HER2 targeted therapy and patients who did not receive HER2 targeted therapy, regardless of the HER2 copy number or clinical stage (Table 1). CONCLUSION: Our preliminary findings suggest that certain patients with HER2 FISH group 3 category breast cancer may not need HER-2 targeted therapy. Larger-scale studies are needed to further evaluate which HER2 FISH group 3 results are more likely to benefit from the HER-2 targeted therapy. Table 1. Response to HER2 targeted therapy in HER2 FISH group 3 breast cancer patients based on HER2 copy numbers and clinical stageHER 2 targeted therapyp-valueGivenNot givenTotal populationGood outcome (n=21)1380.371Bad outcome (n=7)61HER2 copy number < 10Good outcome (n=16)1060.366Bad outcome (n=7)61HER2 copy number ≥ 10Good outcome (n=5)321.000Bad outcome (n=0)00Stage I-IIGood outcome (n=16)970.737Bad outcome (n=3)21Stage III-IVGood outcome (n=5)410.343Bad outcome (n=4)40*Good outcome = No evidence of disease**Bad outcome = Local recurrence, distant metastasis, and/or died of disease Citation Format: Huina Zhang, Rana Ajabnoor, David Hicks, Bradley Turner. Clinicopathologic features and follow-up outcomes of breast cancers with HER2 FISH group 3 Results: A single institution experience [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-48.

INTRODUCTION: Oncotype DX® (ODX) is a multigene assay estimating risk of distant recurrence and chemotherapy benefit in estrogen receptor (ER) positive breast cancer patients. Cost ($4,620.00) impedes its adoption in poorer countries, and the cost is unnecessary in certain patients. TAILORx results suggest that adjuvant endocrine therapy and chemoendocrine therapy had similar efficacy in women with hormone-positive, HER2-negative, node-negative invasive breast carcinomas with an ODX recurrence score < 26. Bhargava et al. and Turner et al. have suggested that a Magee scoreTM or a modified Magee score, respectively, of < 18 will identify patients who are highly likely to have an ODX recurrence score of < 26, and we previously presented data at SABCS 2019 (Abstract P3-07-06) that there is no significant difference in recurrence rate between patients with a modified Magee score of ≤ 18 and patients with an ODX recurrence score < 26 (in preparation for publication). We now present additional recurrence data on patients with a modified Magee score of > 18. METHODS: A total of 301 consecutive patients with ER+ invasive breast cancer from the University of Rochester and the University of Louisville were included in this study, with a mean of 6.6 years of follow-up. All patients had at least 5 years of follow-up (range 5-11 years) except for seven patients , who had a breast cancer recurrence prior to five years. Information on ER, PR, HER-2, Ki-67, Nottingham score , and tumor size were extracted from the pathology report in order to calculate the modified Magee score. Information, on hormone therapy, chemotherapy, radiation therapy, recurrence status, and mortality were extracted from the medical record. For all results, a p-value of < 0.05 was considered significant. RESULTS: 117/301 (39%) patients had a modified Magee score of > 18. The recuurence rate for patients with a modified Magee score > 18 was 11.1%. There was no significant difference in recurrence rate between patients with both a modified Magee score > 18 and ODX recurrence score < 26 compared to patients with both a modified Magee score >18 and ODX recurrence score ≥ 26 (p = 0.547, Table 1). CONCLUSIONS: Patients with a modified Magee score > 18 may be at increased risk for breast cancer recurrence, even if the ODX recurrence score is < 26. Additional studies are necessary to further evaluate these findings. Table 1: Modified Magee score > 18, Oncotype DX recurrence score (ODXRS), and outcomeRecurrenceNo recurrenceModified Magee score > 18 and ODXRS < 26870Modified Magee score > 18 and ODXRS ≥ 26633 Citation Format: Bradley M Turner, Mary Ann Gimenez-Sanders, Ioana Moisini, Huina Zhang, Xi Wang, Linda Schiffhauer, Hani Katerji, Kristin Skinner, Jessica Gooch, Michelle Shayne, Marilyn Ling, Carla Falkson, David Hicks. Are we missing something? Increased recurrence rates in patients with an oncotype DX score < 26 and a modified magee score > 18: A multi-institutional study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-24.

INTRODUCTION: The literature suggests that there is no difference in the Oncotype DX® (ODX) recurrence score between African American (AA) women and Caucasian (CA) women; however, an analysis of clinical outcomes in participants enrolled in the TAILORx trial found that AA women had worse clinical outcomes than CA women, despite similar ODX recurrence scores. The TAILORx trial suggested that chemoendocrine therapy may not be necessary in women with hormone-positive, HER2-negative, node-negative invasive breast carcinomas with an ODX recurrence score < 26. We examine Ki-67 (a marker of tumor proliferation) in relation to an ODX recurrence score of 26 in AA and CA women from two seperate institutions. METHODS: A total of 851 consecutive cases of AA (n = 78) and CA women (n = 773) with ER+ invasive breast cancer and available ODX recurrence scores from the University of Rochester and the University of Louisville were included in this study. For all results, a p-value of < 0.05 was considered significant. RESULTS: Consistent with the previous literatuere, there was no significant difference between the average ODX recurrence score of AA and CA women (p = 0.44, Table 1), and we found no significant difference in average ER, PR, Nottingham score, or tumor size between AA and CA patients; however, the Ki-67 was significantly higher in AA patients with an ODX recurrence score < 26 compared to CA patients with and ODX recurrence score < 26 (p = 0.002, Table 2). This significant difference in Ki-67 was not evident with an ODX recurrence score ≥ 26 (p = 0.98, Table 2). CONCLUSIONS: Our preliminary results suggest that compared to CA women, AA patients are more likely to have a higher Ki-67 if the ODX recurrence score is < 26. AA breast cancer patients with an ODX < 26 are at increased risk for recurrence compared to CA women with an ODX < 26. Ki-67 evaluation is not routinely done as part of the breast cancer workup in many institutions. Ki-67 evaluation may be helpful to help guide treatments decisions in AA patients with and ODX < 26. Additional investigation is warranted. Table 1: Oncotype DX and histopathologic variables for African American and Caucasian womenODXRS* (mean)ER** (mean)PR** (mean)NS***(mean)Ki-67 (mean)****Tumor size (mean)ODXRS < 26 (n) ODXRS ≥ 26 (n)ALL17.6255.3168.96.016.42.2713138AA18.3250.7175.96.321.42.26315CA17.5255.8168.26.015.92.2650123*Oncotype DX recurrence score** modified H-score*** Nottingham score****n = KI-67 available for 777 total patients (650 with ODXRS < 26 and 127 with ODXRS ≥ 26); KI-67 available for 71 AA patients (51 with ODXRS < 26 and 14 with ODXRS ≥ 26); KI-67 available for 706 CA patients (593 with ODXRS < 26 and 113 with ODXRS ≥ 26) Table 2: Oncotype DX < 26 and ≥ 26 and associated histopathologic variables for African American and Caucasian womenODXRS* (mean)ER** (mean)PR** (mean)NS*** (mean)Ki-67 (mean)****Tumor size (mean)ODXRS < 2614.5260.5189.15.813.72.2AA15.4262.8200.76.019.22.3CA14.4260.3188.05.813.12.2ODX ≥ 2633.8228.464.47.330.52.1AA30.3199.871.97.730.41.6CA34.2231.963.57.330.52.2*Oncotype DX recurrence score** modified H-score*** Nottingham score****n = KI-67 available for 777 total patients (650 with ODXRS < 26 and 127 with ODXRS ≥ 26); KI-67 available for 71 AA patients (51 with ODXRS < 26 and 14 with ODXRS ≥ 26); KI-67 available for 706 CA patients (593 with ODXRS < 26 and 113 with ODXRS ≥ 26) Citation Format: Bradley M Turner, Mary Ann Gimenez-Sanders, Huina Zhang, Ioana Moisini, Xi Wang, Linda Schiffhauer, Hani Katerji, Kristin Skinner, Jessica Gooch, Michelle Shayne, Marilyn Ling, Carla Falkson, David Hicks. Are we missing something? Ki-67 evaluation is important in African American women with an oncotype DX score< 26: A multi-institutional study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-18.

Magee Equations™ (ME) are multivariable models that can estimate oncotype DX® recurrence score. One of the equations, Magee Equation 3 (ME3) which utilizes only semi-quantitative receptor results has been shown to provide chemopredictive value in the neoadjuvant setting in a single institutional study. This multi-institutional study (seven institutions contributed cases) was undertaken to examine the validity of ME3 in predicting response to neoadjuvant chemotherapy in estrogen receptor positive, HER2-negative breast cancers. Stage IV cases were excluded. The primary endpoint was the pathologic complete response (pCR) rate in different categories of ME3 scores calculated based on receptor results in the pre-therapy core biopsy. A total of 166 cases met the inclusion criteria. The patient age ranged from 24 to 83 years (median 53 years). The average pre-therapy tumor size was 3.9 cm, and axillary lymph nodes were confirmed positive by pre-therapy core biopsy in 85 of 166 cases (51%). The pCR rate according to ME3 scores was 0% (0 of 64) in ME3 25 to 25 was significantly associated with shorter distant recurrence-free survival and showed a trend for shorter breast cancer-specific survival. The results of this multi-institutional study are similar to previously published data from a single institution (PMID: 28548119) and confirm the chemo-predictive value of ME3 in the neoadjuvant setting. In addition, ME3 may provide prognostic information in patients with residual disease which should be further evaluated.

Breast cancer is the most common invasive cancer in women, with most deaths attributed to metastases. Neoadjuvant chemotherapy (NACT) may be prescribed prior to surgical removal of the tumor for subsets of breast cancer patients but can have diverse undesired and off-target effects, including the increased appearance of the ‘tumor microenvironment of metastasis’, image-based multicellular signatures that are prognostic of breast tumor metastasis. To assess whether NACT can induce changes in two other image-based prognostic/predictive signatures derived from tumor collagen, we quantified second-harmonic generation (SHG) directionality and fiber alignment in formalin-fixed, paraffin-embedded sections of core needle biopsies and primary tumor excisions from 22 human epidermal growth factor receptor 2-overexpressing (HER2+) and 22 triple-negative breast cancers. In both subtypes, we found that SHG directionality (i.e., the forward-to-backward scattering ratio, or F/B) is increased by NACT in the bulk of the tumor, but not the adjacent tumor-stroma interface. Overall collagen fiber alignment is increased by NACT in triple-negative but not HER2+ breast tumors. These results suggest that NACT impacts the collagenous extracellular matrix in a complex and subtype-specific manner, with some prognostic features being unchanged while others are altered in a manner suggestive of a more metastatic phenotype.

The 1983 discovery of a mouse monoclonal antibody—the Ki-67 antibody—that recognized a nuclear antigen present only in proliferating cells represented a seminal discovery for the pathologic assessment of cellular proliferation in breast cancer and other solid tumors. Cellular proliferation is a central determinant of prognosis and response to cytotoxic chemotherapy in patients with breast cancer, and since the discovery of the Ki-67 antibody, Ki-67 has evolved as an important biomarker with both prognostic and predictive potential in breast cancer. Although there is universal recognition among the international guideline recommendations of the value of Ki-67 in breast cancer, recommendations for the actual use of Ki-67 assays in the prognostic and predictive evaluation of breast cancer remain mixed, primarily due to the lack of assay standardization and inconsistent inter-observer and inter-laboratory reproducibility. The treatment of high-risk ER-positive/human epidermal growth factor receptor-2 (HER2) negative breast cancer with the recently FDA-approved drug abemaciclib relies on a quantitative assessment of Ki-67 expression in the treatment decision algorithm. This further reinforces the urgent need for standardization of Ki-67 antibody selection and staining interpretation, which will hopefully lead to multidisciplinary consensus on the use of Ki-67 as a prognostic and predictive marker in breast cancer. The goals of this review are to highlight the historical evolution of Ki-67 in breast cancer, summarize the present literature on Ki-67 in breast cancer, and discuss the evolving literature on the use of Ki-67 as a companion diagnostic biomarker in breast cancer, with consideration for the necessary changes required across pathology practices to help increase the reliability and widespread adoption of Ki-67 as a prognostic and predictive marker for breast cancer in clinical practice.

INTRODUCTION: The recent TAILORx results suggest that adjuvant endocrine therapy and chemoendocrine therapy had similar efficacy in women with hormone-positive, HER2-negative, node-negative invasive breast carcinomas with an Oncotype Dx® recurrence score < 26. Oncotype Dx® is an expensive test (current list price of $4,620.00). Although the Oncotype Dx® test has gained widespread acceptance, cost may be an impediment to its adoption in certain areas of the world, and it may not be the most cost-effective or cost-efficient option in certain subsets of breast cancer patients. Based on a modification of the new Magee equations, we published an algorithmic approach in 283 ER+ cases using an average modified Magee equation (Turner BM, et al. Mod Pathol. 2015;28(7):921-31), and subsequently published a validation of this algorithmic approach in an additional 620 ER+ cases (Cancer Med. 2019 Jun 14. doi: 10.1002/cam4.2323. [Epub ahead of print]), supporting only reflex Oncotype Dx® testing for certain subsets of breast cancer patients. We now have recurrence data and at least five years of outcome data on 310 cases. METHODS: A total of 310 cases with ER+ invasive breast cancer, all with Oncotype Dx® recurrence scores, were included in this study from the University of Rochester and the University of Louisville. The outcome analysis included all patients who had at least five years of follow-up data, and all patients who had a breast cancer recurrence. In order to calculate the average modified Magee score, information on the Nottingham score, ER, PR, Ki-67, HER-2, and tumor size was extracted from the pathology report. RESULTS: 255/310 cases had an Oncotype Dx® < 26, and 4% (10/255) of these cases were associated with a breast cancer recurrence. 187/310 cases had an average modified Magee score ≤ 18, and 184 of these 187 cases (98%) had an Oncotype Dx® recurrence score < 26. Only 2% (n = 4) of these 184 cases were associated with a breast cancer recurrence. On average, the Nottingham score and Ki-67 were lower for cases with an average modified Magee score ≤ 18 compared to cases with an Oncotype Dx® recurrence score < 26 (Table 1). On average, the modified ER and PR H-scores were higher for cases with an average modified Magee score ≤ 18 compared to cases with an Oncotype Dx® recurrence score < 26 (Table 1). CONCLUSION: If the average modified Magee score is ≤ 18, the patient has a low likelihood of recurrence, and the Oncotype Dx® recurrence score will likely be < 26. Given the recent TAILORx findings, we suggest that patients with an average modified Magee score ≤ 18 not be sent out for Oncotype Dx® testing if an Oncotype Dx® recurrence score < 26 is the basis on which the clinician will use to decide whether or not to give chemotherapy. The information needed to use the average modified Magee equation is already generated by many pathology laboratories during the initial assessment of breast cancer, and the Magee equations are free of charge (https://path.upmc.edu/onlineTools/mageeequations.html). Based on the Genomic Health 2017 Annual Report, our data suggests that not sending out cases with an average modified Magee score ≤ 18 would have resulted in a potential cost savings to the health-care system in 2018 of almost 300 million dollars. Table 1: Average Nottingham score, modified ER H-score, modified PR H-score, and Ki-67, with number and percent recurrence for cases with an average modified Magee score ≤ 18 and cases with an Oncotype Dx® recurrence score < 26AVERAGERecurrence Sore typeNNS**ER***PR***Ki-67Recurrence N (%)Average modified Magee score ≤ 18*1875.2262.3235.410.34 (2)Oncotype Dx® recurrence score < 262555.5253.2192.613.610 (4)*98% (184/187) of patients with an average modified Magee score ≤ 18 had an Oncotype Dx® recurrence score < 26**Nottingham score***modified H-score (Turner BM, et al. Mod Pathol. 2015;28(7):921-31) Citation Format: Hasan Khatib, Mary Ann G Sanders, Andrea Breaux, Armen Soukiazian, Nyrie Soukiazian, David Hicks, Bradley M Turner. ER+ patients with an average modified Magee score ≤ 18 have a low likelihood of breast cancer recurrence and a high likelihood of an Oncotype Dx® recurrence score < 26 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-07-06.

INTRODUCTION: Breast cancer is the most prevalent non-skin cancer in women worldwide. Women with metastatic breast cancer to the liver have poor outcomes compared to metastases to other sites, frequently develop resistance to available hormonal and chemotherapeutic agents, and have poor overall survival. Conflicting evidence exists regarding the association of body mass index (BMI) with progression free and overall survival in patients with metastatic breast cancer. METHODS: A single institution retrospective study of women with breast cancer metastasis to the liver was conducted. The pathology reports and medical records from 2011 to 2017 were searched to identify cases with biopsy proven liver metastases. Several factors were considered including breast cancer histologic type and stage at diagnosis; estrogen receptor, progesterone receptor, and HER2-neu amplification status of the initially diagnosed breast cancer as well as the metastatic disease; patient age and ethnicity; BMI (kg/m2) at time of metastasis and at initial diagnosis; and, outcome to include survival after liver metastasis diagnosis. RESULTS: In our population, the BMI ranged from 21.2 kg/m2 to 56.3 kg/m2 (mean 28.4 kg/m2). Eighteen patients (62%) were either overweight or obese. Twenty-one patients (72.4%) were deceased at the time of the study with an average survival of 659 days after liver metastasis diagnosis (Table 1). Patients with a BMI > 30 (obese) had a significantly lower (p Table 1: Mean overall survival in patients with liver metastasisTotalER+ Liver MetastasisHER2+ Liver MetastasisBMI kg/m2N (%)Mean Survival (days)*N (%)Mean Survival (days)*N (%)Mean Survival (days)*Underweight ( 30)7 (33.3)3237 (43.7)3233 (60)315NA: not applicable*p-values Citation Format: Cynthia Reyes Barron, Maricarmen Planas-Silva, David G Hicks, Bradley M Turner. Body mass index and liver metastasis in women with invasive breast carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-08-25.

Management of anal dysplasia relies upon the accurate diagnosis of anal biopsy specimens. As institutions move towards subspecialty signout (SSSO), decisions must be made regarding whether to assign anal biopsies to the gastrointestinal (GI) or gynaecological (GYN) pathology service.We identified 200 archival tissue biopsies of anal mucosa and circulated them among three GI pathologists and three GYN pathologists. Each pathologist separately scored each biopsy as normal, atypical, low-grade squamous intra-epithelial lesion (LSIL) or high-grade squamous intra-epithelial lesion (HSIL). Every case that was called HSIL by at least one pathologist was stained with p16 immunostain and a 'gold standard' interpretation of whether or not a case represented HSIL was made. The GI pathologists agreed on 97 (49%) cases prior to consensus; the GYN pathologists agreed on 33 (17%). The sensitivities of the three GI pathologists in detecting HSIL against the 'gold standard' were 47, 100 and 21% and for the GYN pathologists the sensitivities were 74, 89 and 84%; the sensitivities of the GI and GYN consensus diagnoses were 74% each. The specificities of the three GI pathologists in detecting HSIL were 99, 90 and 100% and for the GYN pathologists the specificities were 99, 92 and 91%; the specificities of both the GI and GYN consensus diagnoses were 100%.A mild to moderate degree of interobserver variability exists in the diagnosis of anal dysplasia among pathologists. Our study indicates the utility of some form of consensus conference, as overall agreement among GI pathologists and among GYN pathologists improved following in-person consensus.

Breast cancer is the most common invasive cancer in women, with most deaths attributed to metastases. Neoadjuvant chemotherapy (NACT) may be prescribed prior to surgical removal of the tumor for subsets of breast cancer patients but can have diverse undesired and off-target effects including increased appearance of the ‘tumor microenvironment of metastasis’ or TMEMs, image-based multicellular signatures which are prognostic of metastasis. In this study we explored whether NACT alters other image-based prognostic/predictive signatures, specifically second-harmonic generation (SHG) directionality, which is indicative of collagen fiber internal structure, as well as the disorganization in collagen fiber alignment. This was performed in paired biopsy/excision samples from 22 patients with HER2 overexpressing invasive ductal carcinoma as well as 22 patients with triple negative breast cancer (TNBC). We found that collagen fiber internal structure, measured using the SHG forward-to-backward-scattered ratio (F/B), is altered in the bulk of the tumor in both tumor types (p = 0.015 and 0.038, respectively), but not the adjacent tumor-stroma interface (p = 0.54 and 0.92, respectively), where F/B is prognostic of metastatic outcome. Overall disorganization in collagen fiber alignment was not significantly changed by NACT in HER2 overexpressing disease (p = 0.41) but was decreased in TNBC (p = 0.0051). These results suggest that NACT alters the collagenous extracellular matrix in diverse ways, with implications for the use of F/B and collagen fiber alignment as prognostic and predictive tools. Citation Format: Edward Brown, Danielle Desa, Robert M Brown, Edward B Brown, Robert L Hill, Bradley M Turner. Second-harmonic generation imaging reveals neoadjuvant chemotherapy-induced changes in breast tumor collagen [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-13.

Objectives: Recently-published phase I clinical trials have demonstrated promising efficacy of novel HER2-targeted therapy in advanced breast cancers with a HER2 immunohistochemistry (IHC) score of 1+, or 2+ with a non-amplified in-situ hybridization (ISH). This has raised the possibility for changing the categories for clinical interpretation of HER2 in breast cancers into: 1) HER2-negative (HER2 IHC score of 0+), 2) HER2-low (IHC score of 1+, or 2+ with a non-amplified ISH), and 3) HER2-positive (HER2 IHC score of 3+ and IHC score of 2+ with amplified ISH). Earlier studies on low HER2-expressing breast cancers focused on differences between HER2 2+ with negative ISH and HER2 0/1+. The aims of this study are to investigate the clinical, pathologic and molecular features of HER2-low breast cancers, in comparison to HER2-negative breast cancers. Methods: 281 breast cancers with HER2 IHC were diagnosed between 04/2020 and 12/2020 at our institution, including 164 HER2-negative, 87 HER2-low, and 30 HER2-positve cases. The clinicopathologic information and molecular subtyping (Agendia BluePrint) results of HER2-low breast cancers were retrospectively collected, and compared to those of HER2-negative cases. A p-value of < 0.05 was considered statistically significant. Results: HER2-low breast cancers accounted for 31% (87/281) of breast cancers in our study population. The majority of these HER2-low cases were clinical stage I-II, with 6 cases (6.9%) being stage IV. Most of the HER2-low cancers had a HER2 IHC score of 1+ (87%, 76/87), a ductal phenotype (82%, 71/87), histologic grades of 1 or 2 (94%, 82/87), were ER positive (94%, 81/86), PR positive (86%, 74/86), and had luminal molecular subtypes (96%, 83/87). Four patients received neoadjuvant chemotherapy and none of them achieved pathologic complete response. Compared to HER2-negative cancers, HER2-low breast cancers showed a lower Ki-67 (p Table 1. Comparison of clinicopathologic parameters and molecular subtypes between HER2-low and HER2-negative breast cancersHER2-Low (n=87)HER2-Negative (n=164)P valueAge (year)66.6264.380.22Tumor size (mm)21.4919.290.49Histologic typeDuctal711390.57Lobular1524Mixed11Histologic grade143640.156239813519pT Stage142880.0452817362Unknown2735PN stage036760.351613210300Unknown2353ER positivity94.2 % (81/86)82.9% (136/64)0.01PR positivity86% (74/86)76.8% (126/164)0.09Ki-67 (%)12.8118.290.03Molecular subtypeLuminal A571050.003Luminal B2630HER210Basal328 Citation Format: Huina Zhang, Hani Katerji, Bradley M Turner, David G Hicks. Clinicopathologic characteristics and molecular profiling of HER2-low breast cancer: A single academic institution experience [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-05-09.

Metastases are the leading cause of breast cancer-related deaths. Tumor metastasis is affected by the microenvironment, including the extracellular matrix (ECM). Fibrillar collagen in the ECM produces an intrinsic optical signal called secondharmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to the collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time. We evaluated the effects of heterogeneity in the tumor ECM on F/B’s prognostic ability. Using SHG imaging we identified two distinct regions within 95 untreated primary tumor excisions: the tumor bulk and surrounding collagenous tumorstroma interface. We found that F/B measured in the tumor-stroma interface, but not tumor bulk, is prognostic of metastasis-free survival time (MFS) using both an intensity threshold selected by a blinded observer and adaptive thresholding. We calculated a 21-gene recurrence score (surrogate OncotypeDX®, or S-ODX) for each patient and using a Random Survival Forest method we found that F/B from the tumor-stroma interface, but not tumor bulk, and S-ODX contribute to predicting MFS in this patient cohort. These results suggest that F/B from the tumor-stroma interface of primary tumor excisions may provide information independent of genomic methods to stratify patients by metastatic risk and identify need for post-operative treatment.

Apoplectic leiomyomas-benign uterine leiomyomas with morphologic changes including hemorrhage, hypercellularity, mitotic activity, nuclear atypia, and even necrosis-can be difficult to distinguish from uterine leiomyosarcomas. Apoplectic leiomyomas have been associated with hormonal therapy; however, the relationship between apoplectic leiomyomas, hormones, and ethnicity has not received much attention in the literature. We evaluated the relationship of hormonal therapy and ethnicity in 869 women with uterine leiomyomas, 136 of which qualified as apoplectic leiomyomas.Apoplectic leiomyomas were observed in 23.3% (49/210) of women exposed to hormonal therapy compared to 13.2% (87/659) of women not exposed to hormonal therapy (p 0.0001). Women taking ethinyl estradiol/norethindrone (Lo-Estrin), leuprolide, and medroxyprogesterone were significantly more likely to have apoplectic leiomyomas compared to women taking other hormonal therapies. Apoplectic leiomyomas were observed in 28.9% (44/152) of African-American women compared to 12.4% (79/639) of Caucasian women (p 0.0001), and this difference remained statistically significant regardless of hormone use. Apoplectic leiomyomas were observed in 22.1% (77/349) of women ≤ 45 years of age compared to 11.3% (59/520) of women 45 years of age (p 0.0001), and this difference remained statistically significant regardless of hormone use.This is the largest study to date examining apoplectic leiomyomas in women on known hormonal therapy compared to women with uterine leiomyomas, but not on hormonal therapy. Information about hormonal therapy, ethnicity, and age can be helpful in the diagnostic interpretation of apoplectic leiomyoma.

Objectives We compared the clinicopathologic features, clinical management, and outcomes of human epidermal growth factor receptor 2 (HER2)–expressing and nonexpressing microinvasive breast carcinomas (MiBC) to explore the significance of HER2 in MiBC. Methods Clinicopathologic and follow-up information of cases with final diagnosis of MiBC with known HER2 status between 2007 and 2019 were analyzed. Results Nineteen (41.3%) HER2-positive (HER2+) and 27 (58.7%) HER2-negative (HER2−) MiBCs were identified. HER2 positivity was likely to be associated with high nuclear grade, presence of tumor-infiltrating lymphocytes, hormonal receptor negativity, and increased Ki-67 in both microinvasive and associated in situ carcinomas. Nodal metastases were found in 2 ER+/HER2− cases (5.3%). One HER2+ case was found to have isolated tumor cells in the axillary node. The majority of patients with HER2+ MiBCs (76.5%) did not receive HER2-targeted therapy. All patients with available follow-up were alive without recurrence or distant metastasis, with a median follow-up of 38 months. Conclusions Similar to the larger size of invasive breast carcinomas, HER2 positivity is associated with high-grade morphologic features in MiBCs. However, HER2 overexpression in MiBCs does not appear to be associated with nodal metastasis or worse outcome in our study cohort. The role of HER2-targeted therapy in this clinical setting merits additional study.

Targeted anti-human epidermal growth factor receptor 2 (HER2) therapy has recently been proven to improve progression-free and overall survival of patients with advanced stage or recurrent endometrial serous carcinoma. To date, no specific pathology HER2 testing or scoring guidelines exist for endometrial cancer. However, based on evidence from the recent successful clinical trial and comprehensive pre-trial pathologic studies, a new set of HER2 scoring criteria have been proposed for endometrial serous carcinoma—distinct from the existing breast and gastric cancer-specific criteria. We present the first study assessing interobserver agreement of HER2 scores using the proposed serous endometrial cancer-specific scoring system. A digitally scanned set of 40 HER2-immunostained slides of endometrial serous carcinoma were sent to seven gynecologic pathologists, who independently assigned HER2 scores for each slide following a brief tutorial. Follow-up fluorescent in situ hybridization (FISH) for HER2 gene amplification was performed on cases with interobserver disagreement when a 2+ HER2 score was assigned by at least one observer. Complete agreement of HER2 scores among all 7 observers was achieved on 15 cases, and all but one case had an agreement by at least 4 observers. The overall agreement was 72.3% (kappa 0.60), 77.5% (kappa 0.65), and 83.3% (kappa 0.65), using four (0 to 3+ ), three (0/1+ , 2+ , 3+ ), or two (0/1+ , 2/3+ ) HER2 scoring categories, respectively. Based on the combination of HER2 immunostaining scores and FISH, the interobserver disagreement may have potentially resulted in a clinically significant difference in HER2 status only in three tumors. We conclude, that the proposed serous endometrial cancer-specific HER2 scoring criteria are reproducible among gynecologic pathologists with moderate to substantial interobserver agreement rates comparable to those of previously reported in breast and gastric carcinomas. Our findings significantly strengthen the foundation for establishing endometrial cancer-specific HER2 scoring guidelines in the future.

Background: We investigate L1CAM expression in ER positive/HER2 negative breast carcinomas. The finding of a potential correlation between high L1CAM expression and recurrent/metastatic disease in luminal A and B breast carcinomas may be helpful for risk stratification and open opportunities for targeted therapies.Methods: 304 cases comprising 152 cases of ER positive, PR positive/negative and HER2 negative recurrent/metastatic breast carcinomas and 152 non-recurrent controls were included. ER, PR, HER-2, Ki-67 status, Nottingham grade, tumor size, tumor stage, number of foci, lymph node status, lymphovascular invasion, phenotype, laterality, age at diagnosis and first distant or local recurrence were recorded. Results: L1CAM positive cases showed increased specificity for recurrence and these patients were significantly younger than L1CAM negative ones. Compared to L1CAM negative recurrent cases, L1CAM positive ones had a noticeably higher Ki-67, tended to be larger and recurred sooner. All L1CAM positive recurrent/metastatic cases were of the luminal B subtype compared to 67.3% of the L1CAM negative cases. Conclusions: L1CAM is highly specific for recurrence in a subset of breast cancer patients and may be associated with more aggressive behavior, particularly in luminal B breast cancers with higher Ki-67 expression. Further investigation about the prognostic value of L1CAM is warranted.

Background: Over-expression of Human Epidermal Growth Factor receptor-2 (HER2) in breast cancer is associated with an aggressive clinical course and poor prognosis. Targeting HER2 over-expression has been shown to be a remarkably effective therapeutic modality in the metastatic, adjuvant and neoadjuvant setting and the pathologic response to neoadjuvant treatment in HER2-positive breast cancer has been shown to be an excellent surrogate for a good outcome. The stromal tumor microenvironment is implicated in fostering tumor growth, facilitating cell migration and ultimately resulting in metastatic disease. Specifically, the collagenous extracellular matrix, which includes fibrillar collagen, has been suggested to play a role in the migration of malignant breast epithelial cells within the surrounding stroma. We have developed a novel methodology which uses an intrinsic optical signature to quantitatively evaluate fibrillar collagen (Burke et al. BMC Cancer 15 (2015): 929). Here, we evaluate the ability of this quantitative methodology to predict the pathologic response after neoadjuvant HER2-targeted treatment as assessed by the Residual Cancer Burden score/class (RCB). This quantitative evaluation in pre-treatment biopsies is then correlated with the pathologic response to treatment in the post-therapy resection. Material and Methods: Clinical pathologic variables for 29 cases of HER2-positive breast cancer that had undergone neoadjuvant chemotherapy plus HER2-targeted therapy were retrieved from the medical record database at URMC, including the post-treatment RCB score and ER/PR/HER2 status. Second harmonic generation (SHG) is an intrinsic optical signal produced by fibrillar collagen. To quantify collagen microstructure in the pre-treatment core biopsy, we used SHG imaging to determine the average forward to backward-light scattering ratio (F/B). The F/B ratio is sensitive to structural properties of collagen fibers. Results: Logistic regression was used to assess the association between F/B and the binary response variable RCB class (0/1 or 2/3). A likelihood ratio test was used to calculate the p-value to test whether the regression coefficient for F/B was zero (i.e. no effect) in the tumor-stromal interface. The average F/B ratio at the leading edge of the tumor stratified by RCB class is shown in Table 1. When evaluated in the bulk of the tumor tissue, F/B was not correlated with RCB status; however, when evaluated at the leading edge of the tumor stromal interface, F/B was significantly correlated with RCB status (p=0.035). Table 1:RCB class and average F/BRCB class (n)Average F/B ± SEM0/1 (19)16.95 ± 1.062/3 (10)12.32 ± 1.84 Conclusions: We have previously shown that the measurement of F/B in the primary tumor after resection is an independent prognostic indicator of metastasis-free survival in breast cancer. Our results in the current study furthers these observations and suggests that the evaluations of the microstructure of collagen fibers by F/B measurement from the pre-treatment biopsy, specifically at the leading edge of the tumor-stroma interface, may be useful for predicting pathologic response to trastuzumab-based neoadjuvant therapy. Further studies in a larger patient cohort are warranted. Citation Format: Desa DE, Turner BM, Buscaglia B, Hill RL, Strawderman RL, Hicks DG, Brown EB. Using multiphoton laser scanning microscopy to assess neoadjuvant therapy outcome in core needle biopsies: A novel methodology [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-12.

Introduction: The recent TAILORx results suggests that adjuvant endocrine therapy and chemoendocrine therapy had similar efficacy in women with hormone–positive, HER2-negative, axillary node–negative breast cancer who had an Oncotype Dx recurrence score (ODXRS) between 11 and 25. These findings, along with updated results that patients with an ODXRS < 11 have a 9-year recurrence rate of only 3%, suggest that certain populations of patients with an ODXRS ≤ 25 may not benefit from additional systemic chemotherapy. Oncotype Dx is an expensive test (current list price of $4,650.00), and cost has been an impediment to its adoption in many centers throughout the United States, and internationally. The test can be performed only at a commercial specialized laboratory. Based on a modification of the new Magee equations (Klein ME, et al. Mod Pathol. 2013;26(5):658–664) we published an algorithm based on 283 patients with ODXRS's (Turner BM, et al. Mod Pathol. 28(7):921-31) that suggested our algorithm offered a less expensive alternative to Oncotype DX testing. We have validated this algorithm in a multi-institutional study on an additional 619 patients with ODXRS's (in preparation for publication). Our validation data, and our data from the original study, also suggests that the average modified Magee equation can reliably predict patients who will have an ODXRS ≤ 25. Methods: 903 cases with an available ODXRS (2006-2018) were identified from the pathology files at the University of Rochester Medical Center (n = 752) and the University of Louisville (n = 151). Information required to calculate the average modified Magee score, which includes estrogen receptor (ER) and progesterone receptor (PR) status, Her-2 status, Nottingham score, tumor size, and Ki-67 were also extracted from the medical record. 78 patients did not have an available Ki-67, leaving 825 patients for inclusion in this study. Results: 478/488 (98%) patients with an average Modified Magee score ≤ 18 had an ODXRS ≤ 25 (only 2% had an ODXRS > 25 - Table 1). 125/337 (37%) patients with an average Modified Magee score >18 had an ODXRS > 25 (Table 1) . Conclusions: The average Modified Magee Score can be helpful in predicting an ODXRS ≤ 25. Patients with an average Modified Magee score ≤ 18 may not need to be sent out for Oncotype Dx testing. The potential cost savings to the health care system would be enormous. Table 1:Average Modified Magee score and risk of an Oncotype Dx recurrence score (ODXRS) > 25Magee scorenAverage ODXRSMinimum ODXRS valueMaximum ODXRS valueCases with ODXRS > 25 [n (%)]≤ 91811.02190 (0)102110.90261 (4.8)113712.10250 (0)126012.00281 (1.7)136412.00230 (0)147012.90271 (1.4)156513.61313 (4.6)165614.12292 (3.6)175214.50272 (3.8)184514.30250 (0)195418.96379 (16.7)204319.30348 (18.6)215921.193715 (25.4)223819.32496 (15.8)232523.383811 (44)242920.83329 (31)252325.0144011 (47.8)> 25-272729.2174620 (74.1)> 27-301634.9225113 (81.3)> 302345.7317823 (100)≤ 1848812.903110 (2)> 1833723.7078125 (37) Citation Format: Turner BM, Sanders MA, Breaux A, Soukiazian A, Soukiazian N, Hicks DG. The average modified Magee score can be helpful in predicting an Oncotype DX recurrence score ≤ 25 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-24.

Introduction: The recent TAILORx results suggest that additional systemic chemotherapy may not be necessary in certain hormone +, HER2 -, node negative breast cancer patients with an Oncotype Dx recurrence score (ODXRS) ≤ 25. ODX is an expensive test (current list price of $4,650.00), and cost has been an impediment to its adoption in many centers throughout the world. Based on a modification of the new Magee equations (Klein ME, et al. Mod Pathol. 26[5]) we published data based on 283 patients with ODXRS's (Turner BM, et al. Mod Pathol. 28[7]), suggesting that the modified Magee equation (MME) offered a less expensive alternative to ODX testing in certain breast cancer patients. We now have outcome data suggesting that the MME along with progesterone receptor (PR), Ki-67, lymph node (LN) status, and lymphovascular invasion (LVI) status can be helpful in predicting which patients with an ODXRS ≤ 25 are more likely to recur. Methods: 248 patients with information on estrogen receptor (ER), PR, Ki-67, Her-2 status, Nottingham score, tumor size, LN status, LVI status, and an available ODXRS (2008-2018) were identified from the pathology files at the University of Rochester Medical Center. Results: All of the patients that recurred had an average modified Magee score (MMS) ≥ 14 (Table 1). Patients with LN involvement (5/43,12% ) or with LVI (5/27,19%) had a higher percentage of recurrence than patients without LN involvement (8/197, 4%) or without LVI (8/216, 4%). Patients that recurred had a significantly (p < 0.05) lower PR and higher Ki-67 than patients in the same risk class that did not recur (Table 2). Neither grade nor ER status was significantly different between patients that recurred and did not recur (Table 2). All of the patients that recurred had at least a lowered PR, higher Ki-67, LN involvement, or LVI, and most had some combination of these variables (Table 1). 8 of the 13 patients that recurred in our population (61.5%) had an ODXRS of ≤ 25. Conclusions: Risk stratification is still important in patients with an ODX score ≤ 25. The MMS along with PR, Ki-67, LN, and LVI status can be helpful in predicting patients with a higher risk of recurrence. Table 1:Recurrence dataODXRSMMSType of therapy*Nodal and LVI status**Nottingham scoreER-H score***PR-H score***Ki-671119.1NONENONE6240180271319.7HNONE530015201521.6HN51209051514HNONE5270210UNKNOWN1615HLVI527018051727.2HB62701601916.3HNONE428545UNKNOWN2424.5CLVI8240105352723.9BNONE7210100352821HN527021252823.4HB528530353128.7HB82701454432.3HNONE9210607021.4****21.4****--5.9****250.7****93.4****31.4*****H = Hormone only;C = Chemo only;B = Both; ** N = Nodal involvement;L = LVI;B = Both; ***modified (Turner BM, et al. Mod Pathol. 2015;28(7):921-31); **** average Table 2:Recurrence data in specific populationsPopulation typeNGradeER*PR*Ki-67≤ 25 no recurrence2165.4249.7185.712.8**≤ 25 recurrence85.5249.4103.325.3***> 25 no recurrence267.3211.776.834.1****> 25 recurrence56.8249.042.542.0* modified (Turner BM, et al. Mod Pathol. 2015;28(7):921-31); ** n = 181; *** n = 6; **** n = 23 Citation Format: Turner BM, Sanders MAG, Soukiazian A, Soukiazian N, Hicks DG. Reconsidering “at risk” criteria for breast cancer recurrence in hormone positive patients: Risk stratification is still important in patients with an Oncotype Dx recurrence score ≤ 25! [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-40.

Purpose The 8th edition of the American Joint Committee on Cancer (AJCC) breast cancer staging system requires histologic grade (GR), estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and stage (assessed by the tumor, node, metastasis classification system). For T1-2 N0, ER+/HER2− tumors, if the 21-gene expression assay is ordered and Oncotype DX (ODX) recurrence score (RS) is 0 to 10, the stage is IA. The purpose of this study was to determine the impact of the ODXRS on staging ER+/HER2− tumors. Materials and Methods This is a retrospective review of ER+/HER2− invasive breast cancer (BC) with ODXRS results from 2 institutions (n = 816) between 2006 and 2018. Stage based on the AJCC 7th and 8th editions, and stage using the 8th edition with and without ODXRS were compared. Significant associations among pathologic parameters and ODX risk groups were determined. Clinical histories were reviewed. Results Nearly half of the patients (43%) had a change in BC stage using the new staging system. Only 4 patients changed stage as a direct result of ODXRS. Influence of ODXRS on staging is limited to T2N0 tumors that are either GR 3 and strongly ER+ and PR+ or GR 1-2 and ER+/PR−. Sixty-one percent of cases of recurrence (11/18) were downstaged using the new staging system. Conclusion ODXRS has little influence on staging, thus supporting the view of the AJCC 8th edition expert panel that ODX is not required for staging. Downstaging of more than half of cases of recurrence suggests that continued refinement of the staging system, as proposed by the expert panel, could be beneficial in this subgroup of patients.

The human epidermal growth factor receptor 2 (HER2) is an important prognostic and predictive biomarker in the breast cancer. The American Society of Clinical Oncology/College of American Pathology (ASCO/CAP) has published HER2 testing guidelines in breast cancer. We herein reviewed the HER2 testing guidelines in breast cancer with a focus on the application of the current guidelines. The continual investigation of HER2 testing in breast cancer has resulted in updates in the HER2 testing guidelines. The current guidelines focus on the uncommon clinical scenarios and emphasize the coordination between immunohistochemistry and in situ hybridization results, in an effort to improve clarity and accuracy. The ASCO/CAP guidelines provide valuable recommendations to ensure the accurate evaluation of HER2 status in breast cancer patients through standardization. Additional studies, particularly those with long-term outcome data are still needed to validate the guideline recommendations, especially the uncommon cases.

Background Clinical assays for the assessment of the human epidermal growth factor receptor-2 (HER2) status in breast cancer include immunohistochemistry (IHC) and in situ hybridization (ISH), both of which have limitations. Recent studies have suggested that a more quantitative approach to the measurement of HER2 protein expression may improve specificity in selecting patients for HER-2 targeted therapy. In the current study, we have used HER2 expression in breast cancer cell lines and clinical samples as a model to explore the potential utility of a novel immunodetection technique, using streptavidin coated Phosphor Integrated Dot fluorescent nanoparticles (PID), which can be quantitatively measured using computer analysis. Methods The expression of HER2 protein in cell lines was evaluated with antibody-binding capacity using fluorescence-activated cell sorting (FACS) for comparison with PID measurements to test for correlations with existing quantitative protein analysis methodologies. Various other analytic validation tests were also performed, including accuracy, precision, sensitivity, robustness and reproducibility. A methods comparison study investigated correlations between PID versus IHC and ISH in clinical samples. Lastly, we measured HER2 protein expression using PID in the pretreatment biopsies from 34 HER2-positive carcinomas that had undergone neoadjuvant trastuzumab-based chemotherapy. Results In the analytic validation, PID HER2 measurements showed a strong linear correlation with FACS analysis in breast cell lines, and demonstrated significant correlations with all aspects of precision, sensitivity, robustness and reproducibility. PID also showed strong correlations with conventional HER2 testing methodologies (IHC and ISH). In the neoadjuvant study, patients with a pathologic complete response (pCR) had a significantly higher PID score compared with patients who did not achieve a pCR (p = 0.011), and was significantly correlated to residual cancer burden (RCB) class (p = 0.026, R2 = 0.9975). Conclusions Analytic testing of PID showed that it may be a viable testing methodology that could offer advantages over other experimental or conventional biomarker diagnostic methodologies. Our data also suggests that PID quantitation of HER2 protein may offer an improvement over conventional HER2 testing in the selection of patients who will be the most likely to benefit from HER2-targeted therapy. Further studies with a larger cohort are warranted.

Cancer therapy is increasingly becoming dependent on the ability to target specific molecular pathways that drive disease progression. Consequently, the identification of specific molecular pathways requires the use of companion diagnostic assays that are accurate and reproducible. This review will discuss the data and concerns surrounding pre-analytic variables in the evaluation of breast cancer tissue. Although ASCO/CAP guidelines offer recommendations for the collection and preservation of diagnostic clinical tissues, standard clinical practice has paid little attention to the suitability of these tissues for further molecular analysis. Current research suggests that alterations in the molecular integrity of tissue during the pre-analytic stage may result in inaccurate results and potentially sub-standard patient care. Threshold recommendations for optimal molecular analyses associated with pre-analytic variables are limited. Future recommendations regarding pre-analytic variables and molecular diagnostics of breast cancer tissue will require additional research on the effects of pre-fixation, fixation, processing, and storage on nucleic acid integrity, comparing fixed material with fresh and/or frozen controls.

We present an updated account of breast cancer treatment and of progress toward "precision" cancer therapy; we focus on new developments in diagnostic molecular pathology and breast cancer that have emerged during the past 2 years. Increasing awareness of new prognostic and predictive methodologies, and introduction of next generation sequencing has increased understanding of both tumor biology and clinical behavior, which offers the possibility of more appropriate therapeutic choices. It remains unclear which of these testing methodologies provides the most informative and cost-effective actionable results for predictive and prognostic pathology. It is likely, however, that an integrated "step-wise" approach that uses the traditional clinical-pathologic paradigms coordinated with molecular characterization of breast tumor tissue, will offer the most comprehensive and cost-effective options for individualized, "precision" therapy for patients with breast cancer.

Background: In 2013, the ASCO/CAP consensus panel published updated guidelines for HER2 testing in breast cancer that modified the definition of HER2 amplification by in situ hybridization (ISH), creating five new prognostic categories (group 1: classic amplified, group 2: monosomy, group 3: co-amplified (polysomy), group 4: equivocal, and group 5: classic non-amplified). Patients determined to be ISH amplified, were considered eligible for HER2-directed therapy. Concern over whether patients from non-classic groups 2-4 would benefit from treatment has led to the recent publication of the 2018 HER2 focused update. This update has modified the criteria for interpreting these ISH categories, recommending that the final diagnosis take into consideration a combination of HER2 immunohistochemistry (IHC) and ISH results. With increased emphasis on the HER2 protein assessment, it has prompted us to quantitatively examine HER2 protein expression in the ISH categories, using two different novel technologies. Materials & Methods: A cohort of 170 cases (URMC) and 102 cases (PSHMC) of invasive breast cancers, which had previously undergone HER2 IHC and ISH testing, were selected for this study. Cases were sorted and categorized into the HER2 ISH categories defined by ASCO/CAP. HER2 protein expression was quantitatively measured in the URMC and PSHMC cohorts using a novel immunodetection methodology (streptavidin-coated Phosphor-Integrated Dot (PID) fluorescent nanoparticles), and a novel dual-antibody, proximity-binding immunoassay (HERmark® Breast Cancer Assay, Monogram Biosciences, South San Francisco, California), respectively. HER2 protein expression was compared to the HER2 FISH and IHC results by ASCO/CAP category. Results: Cases in group 1 had a significantly (p < 0.01) higher average PID/cell and HERmark compared to cases in groups 2-5 (Table 1). Cases in groups 2-4 showed lower quantitative levels of HER2 protein expression, similar to the classic non-amplified cases (group 5). Group 1 was further divided into three subgroups (Table 2): Group A - ISH high-level amplified (ratio > 2, HER2 > 6, CEP17 < 2.7), Group B - amplified with elevated CEP17 (ratio > 2, CEP17 > 2.7), and Group C - low-level amplified (ratio > 2, HER2 > 4 and < 6). Group A and B had a significantly (p < 0.01) higher average PID/cell and HERmark compared to Group C. Group C was more comparable to cases in groups 2-5 (Table 1). Conclusion: Our results suggest that quantitative assessment of HER2 protein expression may help to further classify cases for HER2 status for targeted therapy, supporting the 2018 ASCO/CAP recommendation that non-classic ISH results might be resolved by evaluating protein expression. Follow up studies with a larger patient cohort and dual quantitative assessment are warranted. Average PID/cell and HERmark in ASCO category groupsASCO category groupN (URMC)PID/cell (URMC)*N (PSHMC)HERmark (PSHMC)*18888.07761.521011.20N/A32016.0213.84238.5315.95296.3208.3*averageTable 2:Average PID/cell and HERmark in subgroups of Group 1SubgroupN (URMC)PID/cell (URMC)*N (PSHMC)HERmark (PSHMC)*A24157.66465.7B34101.61044.1C3016.9329.8*average Citation Format: Buscaglia B, Turner B, Goda H, Huang W, Leitzel K, Natori T, Nakano Y, Okada H, Sperinde J, Ali S, Vasekar M, D'Aguiar M, McMahon L, Henry J, Lipton A, Hicks D. ASCO/CAP human epidermal growth factor receptor-2 (HER2) in situ hybridization (ISH) categories evaluated by quantitative HER2 protein diagnostic methodologies: A comparative analysis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-03-02.

Objectives To investigate human epidermal growth factor receptor 2 (HER2)-positive nonpleomorphic invasive lobular carcinoma (ILC), which has rarely been addressed. Methods Clinicopathologic characteristics and follow-up of HER2-positive nonpleomorphic ILCs were collected and compared to those of HER2-negative counterparts. Results Twenty-one cases of HER2-positive nonpleomorphic ILCs were identified, 6.3% of the study population. Compared to HER2-negative nonpleomorphic ILC, patients with HER2 positivity were older (P < .05), likely to be hormonal receptor negative (P < .01), and had higher histologic grade and angiolymphatic invasion (P < .01). HER2 positivity in nonpleomorphic ILCs was associated with higher recurrence/metastasis with hazard ratio of 2.03 (P < .05). No patient who received neoadjuvant therapy achieved pathologic complete response, and HER2-targeted therapy tended to reduce recurrence/metastasis in patients with HER2-positive nonpleomorphic ILC. Conclusions Our results highlight the existence of HER2 positivity in nonpleomorphic ILCs and reinforce that HER2 is associated with worse prognosis in nonpleomorphic ILC.

Objectives. We recently suggested that increased intramural pressure may often explain pain and/or bleeding. Hysterectomies for bleeding tend to have outward bulges and endometrial vascular ectasia, while hysterectomies for pain tend to have deflection of pressure inward by subserosal ridges, which promote inner myometrial elastosis (IME). Study design. We analyzed causes of increased intramural pressure in 58 hysterectomies for pain and/or bleeding, excluding clinically fibroid uteri and prolapsed uteri. Postfixation photographs were used to avoid missing grossly obvious myometrial hyperplasia (MMH). Results. The most common cause of increased intramural pressure was grossly obvious MMH in 40/58 cases (69%). Other causes included clinically occult myomas (3 cases), adenomyosis (6 cases), and multifactorial causes (7 cases). Hysterectomies for bleeding weighed more than hysterectomies for pain ( P = .035). Hysterectomies for pain had more IME than hysterectomies for bleeding ( P = .029). Conclusions. When subserosal ridges deflect pressure inward, bulky MMH may cause pelvic pain and IME, but when they do not, bulkier MMH in heavier uteri may lead to both outward bulges and abnormal bleeding from endometrial vascular ectasia.

Neoadjuvant chemotherapy (NACT) is routinely administered to subsets of breast cancer patients, including triple negative (TN) or human epidermal growth factor receptor 2-positive (HER2+) cancers. After NACT and subsequent surgical resection, 5% to 30% of patients have no residual invasive carcinoma, termed pathological complete response. Unfortunately, many patients experience little-to-no response after NACT and unnecessarily suffer its side effects. Methods are needed to predict an individual patient’s response to NACT. Core needle biopsies, taken before NACT, consist of tumor cells and the surrounding extracellular matrix. We performed second-harmonic generation (SHG) imaging of fibrillar collagen in core needle biopsy sections as a possible predictor of response to NACT. The ratio of forward-to-backward scattering (F/B) SHG was assessed in the “tumor bulk” and “tumor–host interface” in HER2+ and TN core needle biopsy sections. Patient response was classified post-treatment using the Residual Cancer Burden (RCB) score. In HER2+ biopsies, RCB class was associated with F/B derived from the tumor–stromal interface, but not tumor bulk. F/B was not associated with RCB class in TN biopsies. These findings suggest that F/B from needle biopsy sections may be a useful predictor of which patients will respond favorably to NACT, with the potential to help reduce overtreatment.

It has been suggested that impaired venous drainage and endometrial vascular ectasia (EMVE), secondary to increased intramural pressure, explains abnormal bleeding in fibroid uteri. Striking EMVE with extravasated red blood cells (ecchymosis) has also been seen in uteri with grossly obvious myometrial hyperplasia (MMH), suggesting that increased intramural pressure can cause EMVE in the absence of fibroids. EMVE with MMH may explain the century old association of clinically enlarged uteri with abnormal bleeding, and this same mechanism may be operative in myopathic uteri with grossly obvious adenomyosis. EMVE with associated thrombosis, ecchymosis, and/or stromal breakdown is commonly seen in random sections of hysterectomies for bleeding. EMVE may also be associated with endothelial hyperplasia, consistent with a reaction to endothelial injury due to impaired venous drainage. This further supports the theory that EMVE bleeds when thrombosis occurs, due to Virchow's Triad (stasis, endothelial injury, and hypercoagulability). EMVE may be "the lesion for which surgery was performed" in hysterectomies with otherwise unexplained bleeding.

Second harmonic generation (SHG) is an intrinsic optical signal that can be generated from fibrillar collagen. The directionality of SHG emission is influenced by the diameter and spacing of collagen fibrils, and the disorder in their packing within collagen fibers. One measure of SHG directionality is the ratio of forward- to backward-scattered SHG, or “F/B”. F/B has been used to assess healthy vs diseased tissue in breast, ovarian, and lung cancer, and is an independent prognostic indicator of metastasis free survival time in ER+, lymph node-negative (N0), invasive ductal carcinoma (IDC). Here we assess the heterogeneity in F/B within tumor sections from ER+ N0 IDC, and its effect on the use of F/B for metastatic risk prediction. We find that F/B of tumor collagen varies between the tumor/host interface and the more cellular tumor bulk (p Citation Format: Edward Brown, Danielle Desa, Wencheng Wu, Robert Hill, John Martens, Robert Strawderman, Bradley Turner. Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-08.

The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked), has been comprehensively studied by in vitro immunohistochemical (IHC) staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by micro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG3 (control), was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untargetable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper "Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis" (Neoplasia 19: 716-733, 2017), and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124I- JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need.

Oncotype DX (Genomic Health, Redwood City, CA, USA, current list price $4,350.00) is a multigene quantitative reverse transcription-polymerase chain reaction-based assay that estimates the risk of distant recurrence and predicts chemotherapy benefit for patients with estrogen receptor (ER)-positive breast cancers. Studies have suggested that standard histologic variables can provide similar information. Klein and Dabbs et al have shown that Oncotype DX recurrence scores can be estimated by incorporating standard histologic variables into equations (Magee equations). Using a simple modification of the Magee equation, we predict the Oncotype DX recurrence score in an independent set of 283 cases. The Pearson correlation coefficient (r) for the Oncotype DX and average modified Magee recurrence scores was 0.6644 (n=283; P0.0001). 100% of cases with an average modified Magee recurrence score30 (n=8) or an average modified Magee recurrence score9 (with an available Ki-67, n=5) would have been correctly predicted to have a high or low Oncotype DX recurrence score, respectively. 86% (38/44) of cases with an average modified Magee recurrence score≤12, and 89% (34/38) of low grade tumors (NS6) with an ER and PR≥150, and a Ki-6710%, would have been correctly predicted to have a low Oncotype DX recurrence score. Using an algorithmic approach to eliminate high and low risk cases, between 5% and 23% of cases would potentially not have been sent by our institution for Oncotype DX testing, creating a potential cost savings between $56,550.00 and $282,750.00. The modified Magee recurrence score along with histologic criteria may be a cost-effective alternative to the Oncotype DX in risk stratifying certain breast cancer patients. The information needed is already generated by many pathology laboratories during the initial assessment of primary breast cancer, and the equations are free.

Clinical decisions regarding the suitability of adjuvant systemic therapy for individual patients with breast cancer depends on comprehensive assessment of the underlying biology of each patient's tumor. The previous clinical-pathologic paradigm for treatment, which had been used for decades, now has been augmented by significant advances in molecular analysis of breast tumor tissue samples. Molecular testing has the potential to understand better both tumor biology and clinical behavior, which enables more appropriate therapy choices to be made. We review the rapid evolution in profiling breast cancer tissues, and discuss the current evidence for clinical use of this information and how the emerging molecular paradigm can be integrated into the clinical-pathologic context as we progress toward "precision" therapy for patients with breast cancer and other solid tumors.

INTRODUCTION: Oncotype DX (ODX) (Genomic Health, Redwood City, CA, USA) is a multigene quantitative reverse transcription-polymerase chain reaction-based assay used to estimate the risk of distant recurrence for ER-positive, node negative-breast cancer patients. The quoted list price for ODX is $4.175.00. ODX reports a recurrence score (RS) that is divided into low (< 18), intermediate (18-30), and high risk (≥ 30). The ODX RS has been shown to be prognostic and predict chemotherapy benefit in receptor positive breast cancer patients. The RS most highly weights genes related to proliferation (including Ki-67), HER-2, ER and PR. Klein et al. have previously shown that standard histological variables, in combination with semiquantitative ER, PR, HER-2, and Ki-67 results, can provide information similar to that found in the ODX RS using equations derived by linear regression analysis (Magee equations). In the current study, we have used a modification of these equations to validate their use in a cohort of breast cancer cases that were sent for ODX from our institution. MATERIALS AND METHODS: 233 cases (2009-present) with available ODX RS's (208 of these with Ki-67 results) were obtained from the pathology files at the University of Rochester Medical Center (URMC), and the histopathologic variables were extracted from the reports. Estimated RS's (URMC RS) were calculated by averaging results from the three published Magee equations, with a modification of the H-score for ER and PR. We estimated the H-score by multiplying the average intensity of staining by the% positive tumor cells. URMC RS's were compared with their paired ODX RS’s. RESULTS: The table shows the correlation between the ODX RS and URMC RS. 100% of cases with a high URMC RS also had a high ODX RS. There were no two step discordant results between a URMC RS and an ODX RS. A URMC RS correlated with an ODX RS of high, intermediate, and low in 7/12 (58%), 48/79 (61%) and 97/117 (83%) cases, respectively. Most of the discordant cases involved the intermediate/low groups, and the higher RS's in the discordant pairs were consistently close to the lower end cutoff point.The URMC RS and ODX RS would have resulted in similar treatment in 100% of patients. Comparison of ODX RS and URMC RS; n = number of cases (URMC median, URMC range) ODX RS ⇒URMC RS ⇓HIGHINTERMEDIATELOWHIGH7 (35,32-40)00INTERMEDIATE5 (25,22-28)48 (22,18-30)20 (20,18-27)LOW031 (15,9-18)97 (14,7-18) CONCLUSION: The modified Magee equations provide similar information as the ODX RS using information already generated by many laboratories, and may be a more cost-effective alternative than ODX in identifying breast cancer patients who might benefit from chemotherapy, or be spared its potential toxicity. If the estimated RS is clearly high or low, it is predictive of the ODX RS with a high degree of certainty. In our population a potential cost of $556,875.00 might have been avoided using the estimated RS. The estimated RS should not be dramatically different from the ODX RS. If there is a significant difference, this should be thoroughly investigated before decisions on adjuvant therapy are made. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-12.