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.