Abstract 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.

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 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. 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.

Abstract 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.

Abstract Recent reporting of the 9 year follow-up for the TAILORx trial suggests that there may be no benefit with adjuvant chemotherapy for ER +, HER2 -, N(0) breast cancer patients with a Oncotype DX® (ODX) recurrence score (RS) <26. Since endocrine therapy for this group of patients who comply with treatment still results in distant recurrence (rMBC) in 3% and 5% of the ODX low and ODX intermediate risk groups at 9 years, respectively, we are motivated to help find early treatments for these patients by identifying their recurrence risk at diagnosis with improved risk stratification. Methods: Optical Prediction of Time Interval to Metastasis (OPTIM), a novel assay, prognostic for rMBC, is based on an intrinsic optical signature from collagen, derived from the average of point by point ratios of forward to backward (F/B) second harmonic generation (SHG) light scatter that is sensitive to form and structure of fibrillar collagen in the extracellular matrix of archival tissue microarray samples. (Burke et al. BMC Cancer 15 (2015): 929). The 125 patients in this cohort were part of a clinical trial, looking for genomic predictors of rMBC in untreated patients, so we were able to calculate a surrogate 21-gene RT-PCR assay (S-ODX) value based on gene expression data available through NCBI GEO database (Gyorffy et al. Breast Cancer Res Treat (2012) 132:1025). We analyzed these patient's rMBC outcomes using logistic regression and Kaplan-Meier (KM) analysis. Results: OPTIM alone stratified at 2.5X relative risk (RR) between quartiles Q1 and Q4, similar to S-ODX low vs high recurrence score (RS) groups (from TAILORx Trial) with 2.8X RR. Using quartiles of OPTIM vs S-ODX together we stratify patients to recurrence risk (rMBC/at Risk), with an improved risk stratification of 5X RR in the RS<26 low risk groups. OPTIM Quartiles vs RS Risk Groups in TAILORx TrialS-ODX →High (RS>25)Intermediate (RS 11-25)Low (RS <11)AllOPTIM↓↓↓↓Q17/97/12*5/1019/31*Q25/95/142/812/31Q38/12***1/81/11***10/31Q46/10**2/17*0/5**8/32*All26/40***15/518/34***49/125Recurrence at 10 years by KM analysis *p<0.05, **p<0.005, ***p<.0005 Combining S-ODX with OPTIM, low (L) or high (H) risk by assay, shows that they are independent and complementary. Notably 68%=85/125 are classified L by S-ODX (RS<26) and OPTIM effectively reclassifies H and L, and when combined with S-ODX H identifies 92%=45/49 of all rMBC at 10 years without treatment. Risk stratification improves to 6.8X RR comparing highest risk HH 66.7%=12/18 to lowest risk LL 9.8%=4/41. Distant Recurrence Identified by High Risk Group of Each AssayS-ODX AssayHHLLOPTIM AssayHLHLrMBC (total=49)1214194At Risk (total n=125)18224441rMBC at 10 yrs. S-ODX RS>25=H, RS<26=L; OPTIM Q1&Q2=H, Q3&Q4=L Conclusion: OPTIM as an independent prognostic optical bio-marker from collagen in intact tissue. Combination of OPTIM with the Oncotype DX® assay may produce a continuous risk estimator with higher dynamic range than either assay alone and will be the focus of future study, especially in a treated population, to determine if OPTIM might also predict response to treatment. Citation Format: Hill RL, Perry SW, Salzman P, Turner BM, Hicks DG, Brown EB. Optical Prediction of Time Interval to Metastasis (OPTIM): A rapid nondestructive optical assay applied to tissue microarray samples identifying high risk of distant recurrence in the lowest risk groups defined by the TAILORx trial [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 P6-09-05.

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.

Abstract Introduction: Neoadjuvant chemotherapy is used in locally advanced breast cancer to downstage the tumor, facilitating surgical management. Oncotype DX (ODX) is used to estimate the risk of distant recurrence for ER-positive breast cancers, allowing selected patients to avoid the toxicity of chemotherapy. ODX is often not possible on the small core biopsy samples. Klein et al. have shown that standard histological variables, combined with semiquantitative ER, PR, HER-2, and Ki-67 results, can provide information similar to that with ODX, using equations derived by linear regression analysis (Magee equations). We applied a modification of these equations to pretreatment core biopsies in women who received neoadjuvant chemotherapy to determine if the risk scores were predictive of pathologic response. Methods: 25 patients who received chemotherapy for receptor positive locally advanced(21), inflammatory(3), or metastatic(1) breast cancer followed by surgical treatment of the primary site were identified from a prospective breast cancer database. Pretreatment core biopsies were reviewed by a breast pathologist and Nottingham grade, ER and PR status (% of cells staining and intensity of staining), and Her-2 status by IHC and/or FISH were recorded. Clinical tumor size was defined as the average of sizes derived from mammogram, ultrasound, MRI, PET-CT and clinical breast examination. Using these data in a modified Magee equation, the patient's recurrence score was calculated. 0-18 was considered low risk (LR), >18-<30 was considered intermediate risk (IR), and ≥30 was considered high risk (HR). Resection specimens were reviewed to define pathologic response. A good pathologic response to chemotherapy was defined as a complete pathologic response (3 cases), near complete response (2), or a response with one or more of the following; reduction in the post-treatment size of the tumor by greater than 50% compared with pretreatment imaging, a significant reduction in tumor cellularity in the tumor bed, and an inflammatory lymphohistiocytic infiltrate with tumor necrosis (6 cases). For the remaining 14 cases, the response was defined as poor (no histopathologic evidence of response to treatment). Risk scores were compared between good and poor responders using T-Test. Comparison between risk groups (HR vs IR vs LR) were made using Chi Square analysis. Results: Magee scores ranged from 13.8-41.6 (mean 27.4) and were significantly lower in the poor responders (mean = 23, range 13.8-41.6) compared to the good responders (mean = 33, range 22-41.3, p = 0.003). Table 1 shows the distribution of response by Risk Group (p = 0.018). Table 1: Response by Risk CategoryMagee Risk GroupLRIRHRPoor Response563Good Response038 73% of patients with high risk Magee scores had a good response to chemotherapy, compared to 21% of patient with low or intermediate scores (p = 0.01). Conclusions: Modified Magee equations applied to pretreatment core biopsies seem to predict pathologic response to neoadjuvant chemotherapy. Use of these equations to assign risk scores may be a useful tool in deciding which ER positive breast cancer patients are likely to benefit from preoperative chemotherapy for cytoreduction, and who should go directly to surgery. These findings need to be validated in larger studies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-37.

Abstract 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.

Abstract BACKGROUND: Immunohistochemical (IHC) analyses of protein expression have recently been used as surrogate assays for the molecular classification of breast cancers. Specifically, ER+/PR+/HER2- tumors are typically considered luminal-type breast cancers, ER-/PR-/HER2+ tumors equate to HER2 over-expressing tumors, whereas ER-/PR-/HER2- (triple negative, TN) tumors are basal-like breast cancers. A newly available cocktail of antibodies called ADH-5 allows for the simultaneous evaluation of both luminal and basal keratins. In this study, we examine the expression of luminal and basal keratins in breast cancers stained with ADH-5, particularly in a subgroup of basal keratin positive tumors. METHODS: All invasive breast cancers from 12/2009-6/2010 in a single pathology laboratory were studied by ADH-5 antibody cocktail consisting of two luminal keratin (LK) markers (CK7 and CK18), and two basal keratin (BK) markers (CK5 and CK14), as well as a myoepithelial marker, P63 (Biocare, Concord, CA). ER, PR, HER2 and Ki-67 were also evaluated by IHC and scored in all tumors. The group included 81 breast cancers and 3 metastatic tumors from the breast. ADH-5 staining was evaluated in areas of invasive carcinoma, and intensity and proportion of staining by BK and LK were scored. Mean statistics and ratios were evaluated and compared for each of the scored stains using the student t-test (stt) and chi squared analysis (cs), respectively. RESULTS: Of the 84 cancers, 20 (24%) showed some staining for BK, and 63 (75%) showed only LK staining. One case (1%) showed no staining for either BK or LK. Of the BK+ cases, 19/20 (95%) showed some degree of LK staining as well. Only one BK+ tumor lacked luminal keratin positivity (5%). Co-expression both BK and LK was seen in the same cells, though many cells were more strongly positive for one keratin type. The BK+ tumors were significantly more likely to be TN (p = 0.5, cs), and all TN tumors showed some BK staining. Four ER+/PR+/HER2- tumors expressed low levels of BK; two additional ER+/PR+/HER2+ tumors also expressed BK. ER-, PR-and TN tumors tended to show a significantly greater proportion of BK staining and decreased amount of luminal staining than ER+, PR+ and non-TN tumors. DISCUSSION: ADH-5 stain demonstrates co-expression of luminal keratins in basal keratin positive breast cancers, including all triple negative breast cancers in our study. In addition, some ER+/PR+/HER2- tumors and ER+/PR+/HER2+ (considered luminal A and B, respectively, by current usage of ER/PR/HER2 as surrogate markers) actually express basal keratins, bringing up the issue of whether these tumors should be considered luminal or basal-like. The ADH-5 stain may be helpful as a surrogate molecular marker for the classification of breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-15.