Hoskin, Tanya L.

Abstract Introduction: We used micro-computed tomography (microCT), a high-resolution imaging option, to detect and characterize microcalcifications (MCs) in three dimensions within pathology blocks of benign and malignant breast tissues. Methods: A set of 44 formalin-fixed, paraffin-embedded breast tissue blocks were assessed from n=22 women at two time points: benign breast biopsy and subsequent DCIS or DCIS with invasive disease breast cancer. Blocks were scanned using the Bruker Skyscan 1276 microCT at a resolution of 10 µm. SkyScan analysis CTAn software was utilized to prepare and analyze morphological parameters and multiple MCs contained within each block were averaged. Paired morphological parameters were compared between the benign and subsequent cancer samples within women using Wilcoxon signed-rank tests. Results: Median age of patients was 53 years (range 37-77 years) at initial benign biopsy and developed breast cancer at median of 5.6 years (range: 2.3-12.8 years) later. The initial benign biopsies were for proliferative disease without atypia in the majority (59%), while 23% had non-proliferative lesions and 18% atypical hyperplasia. The average structural model index was consistent with a near rod-like shape (median 2.99, range: 2.71-3.29). Paired comparisons between benign and cancer blocks showed that the average surface area of MCs was significantly higher in the cancer samples (p=0.0467), as was the average volume of MCs (p=0.0298) (Table 1). Conclusions: Preliminarily, we demonstrate that microCT of routinely prepared pathology blocks of breast tissues may be useful in characterizing the 3-D morphometry of MCs with suggestive differences in benign and cancer tissues. Given that MCs are a sentinel radiologic marker of DCIS but are common in benign breast disease, developing improved approaches for classifying MCs may have value in breast cancer screening and management. Table 1. Comparison of MC parameters in biopsy tissues (IQR = Interquartile Range). Benign sample from women who later developed breast cancer Breast cancer after prior benign sample Paired Differences (Cancer - Benign) Wilcoxon signed-rank p value (N=22) (N=22) (N=22) Number of Objects (Median (IQR)) 43.5 (14, 789) 69.5 (33, 289) -6 (-139, 67) 0.6830 Average Surface Area (mm2)(Median (IQR)) 0.0225 (0.0134, 0.0393) 0.0342 (0.0185, 0.0656) 0.0113 (-0.0027, 0.0322) 0.0467 Average Volume (mm3) (Median (IQR)) 0.0003 (0.0001, 0.0007) 0.0007 (0.0003, 0.0022) 0.0003 (0.000002, 0.0020) 0.0298 Average Surface Area/Volume (mm−1)(Median (IQR)) 214.92 (162.25, 328.39) 199.55 (151.89, 230.52) -20.85 (-86.05, 27.04) 0.1433 Average Structural Thickness (mm)(Median (IQR)) 0.0341(0.0196, 0.0446) 0.0395 (0.0336, 0.0505) 0.0074 (-0.0062, 0.0153) 0.1173 Average Structural Model Index (Median (IQR)) 2.9758 (2.8465, 3.1175) 3.0026 (2.9281, 3.0927) 0.0075 (-0.1073, 0.1381) 0.7656 Citation Format: Sarah E. Schrup, Thomas de Bel, Tanya Hoskin, Teresa Allers, Stacey Winham, Derek Radisky, Laura Pacheco-Spann, Lisa Seymour, Amy Degnim, Mark Sherman. High resolution microCT to analyze the 3D morphology of microcalcifications in benign breast disease and breast cancer biopsy tissues. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3587.

Abstract Purpose: To evaluate the impact of trimodality treatment versus monotherapy or dual therapy for radiation-associated angiosarcoma of the breast (RAASB) after prior breast cancer treatment. Experimental Design: With Institutional Review Board approval, we identified patients diagnosed with RAASB and abstracted data on disease presentation, treatment, and oncologic outcomes. Trimodality therapy included (i) taxane induction, (ii) concurrent taxane/radiation, and then (iii) surgical resection with wide margins. Results: A total of 38 patients (median age 69 years) met inclusion criteria. Sixteen received trimodality therapy and 22 monotherapy/dual therapy. Skin involvement and disease extent were similar in both groups. All trimodality patients required reconstructive procedures for wound closure/coverage, compared with 48% of monotherapy/dual therapy patients (P < 0.001). Twelve of 16 (75%) patients receiving trimodality therapy had a pathologic complete response (pCR). With median follow-up of 5.6 years, none had local recurrence, 1 patient (6%) had distant recurrence, and no patients died. Among 22 patients in the monotherapy/dual therapy group, 10 (45%) had local recurrence, 8 (36%) had distant recurrence, and 7 (32%) died of disease. Trimodality therapy demonstrated significantly better 5-year recurrence-free survival [RFS; 93.8% vs. 42.9%; P = 0.004; HR, 7.6 (95% confidence interval, CI: 1.3–44.2)]. Combining all patients with RAASB regardless of treatment, local recurrence was associated with subsequent distant recurrence (HR, 9.0; P = 0.002); distant recurrence developed in 3 of 28 (11%) patients without local recurrence compared with 6 of 10 (60%) with local recurrence. The trimodality group had more surgical complications that required reoperation or prolonged healing. Conclusions: Trimodality therapy for RAASB was more toxic but is promising, with a high rate of pCR, durable local control, and improved RFS.

Abstract Background: The role of chemotherapy in node positive (N+) luminal breast cancer (BC) is often debated, given low total pathologic complete response (pCR) rates following neoadjuvant chemotherapy (NAC) and discrepancy in adjuvant chemotherapy benefit. A prior single institution study of cN+ luminal BC showed that pts age < 50 and tumor Ki-67 ≥ 20% had high nodal pCR (> 35%). This study’s goals were 1) to validate Ki-67 and age in relation to nodal pCR and 2) evaluate the prognostic impact of nodal pCR on overall survival (OS). Methods: We queried the National Cancer Database 2010-2019 for pts with cN+ ER+/HER2- BC treated with NAC and surgery. Breast pCR was defined as ypT0/ypTis and nodal pCR as ypN0/ypN0i+. Ki-67 was available in 2018 & 2019 only and was used to evaluate Ki-67 and nodal pCR. 2010-2018 data were used to evaluate nodal pCR and OS. OS was analyzed using multivariable Cox proportional hazards regression. Results: In 2018-2019, 4,801 pts were identified and 2,473 (51.5%) had Ki-67 available. Nodal pCR was 23.7% and was higher in pts < 50 years old (28.1% vs 21.1%) and in those with Ki67 ≥ 20% (28.4% vs 12.7%), both p < 0.001. Pts < 50 with Ki67 ≥ 20% had the highest nodal pCR at 31.7%, followed by age ≥ 50 with Ki67 ≥ 20% at 26.3%. With Ki67 < 20%, nodal pCR was 15.4% (in age < 50) and 11.3% (in age ≥ 50). From 2010-2018, we identified 20,084 cN+ ER+/HER2- BC pts treated with NAC. Total pCR was 7.4%, 14.3% had nodal pCR only, 3.8% had breast pCR only, and 74.5% had residual disease in breast and nodes. OS at 5 years was 79.1% and varied by NAC response: 90.8% with total pCR, 83.8% with nodal pCR only, 80.7% with breast pCR only, and 76.9% with residual disease in breast and nodes. Specifically nodal pCR (with or without breast pCR) was seen in 22.0% and was associated with 5-year OS rate of 86.4% compared to 77.1% without nodal pCR, p < 0.001. On multivariable analysis adjusted for other clinical and treatment factors, nodal pCR was associated with better OS (adjusted HR 0.56, 95% CI: 0.50-0.61, p < 0.001) in all ages combined and within both the age < 50 and age ≥ 50 subgroups (see Table). In a subgroup of pts approximating RxPonder entry criteria (defined as cT1-3, N1, Grade I or II, ER+/PR+), results were consistent with the overall cohort: nodal pCR varied by both age (17.5% in age < 50 and 13.6% in age ≥ 50, p < 0.001) and by Ki67 ≥ 20% vs < 20% (16.8% vs 7.9%, p < 0.001) and nodal pCR remained prognostic for OS with adjusted HR 0.63 (95% CI: 0.50-0.81, p < 0.001). Conclusion: In cN+ ER+/HER2- BC treated with NAC, nodal pCR is more common in pts< 50 and those with high Ki-67 and is highly prognostic for OS. These data strongly suggest that NAC chemotherapy benefit should not be evaluated using total pCR rates in isolation, but for N+ pts to also consider nodal response. Given that nodal pCR is highly prognostic for OS, future neoadjuvant strategies should consider nodal pCR as a potential intermediate biomarker for long term survival. Multivariable analysis of factors associated with overall survival, including the adjusted effect of nodal pCR Citation Format: Dan Moldoveanu, Matthew P. Goetz, Tanya L. Hoskin, Courtney N. Day, Judy C. Boughey. Age, Ki-67, Nodal pCR and overall survival following Neoadjuvant Chemotherapy for Node Positive ER+/Her2- Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-22.

Abstract Background: Adjuvant endocrine therapy (ET) in ER+ breast cancer (BC) reduces local, distant, and contralateral BC events and improves overall survival (OS). Furthermore, decreased adherence or omission of ET increases the risk of death. However, in ER+ pts with early-stage BC treated with NAC who have a pathologic complete response (pCR), the importance of adjuvant ET may be called into question. We sought to examine the impact of ET omission on the survival of pts with ER+ BC treated with NAC, according to pCR vs residual disease. Methods: We queried the National Cancer Database (NCDB) 2010-2018 for female pts with stage I-III ER+ BC treated with NAC followed by surgery. pCR was defined as ypT0/ypTis, ypN0. The percent receiving adjuvant ET and the impact of adjuvant ET omission on overall survival (OS) in patients with and without pCR were assessed separately based on HER2 expression. OS was analyzed with adjuvant ET as a time-dependent covariate using Cox proportional hazards regression. Results: We identified 34,394 pts treated with NAC for ER+ BC (28,434 ER+/HER2-, 5960 ER+/HER2+). Pts with ER+/HER2+ BC were less likely than pts with ER+/HER2- BC to have received adjuvant ET (61.6% vs 88.8%, p< 0.001). Overall, 4505 (13.1%) had pCR (9.1% of ER+/HER2- and 32.0% of ER+/HER2+). Within each subtype, pts with pCR were significantly less likely to start adjuvant ET after surgery than pts with residual disease (78.4% vs 89.8% for ER+/HER2- and 46.5% vs 68.7% for ER+/HER2+, each p< 0.001), Table 1. Regarding those with residual disease, pts with ER+/HER2+ BC were less likely than ER+/HER2- BC to receive adjuvant ET (68.7% vs 89.8%, p< 0.001). Median follow-up was 4.4 years. Among pts with pCR, 5-year OS was 93.2% (95% CI: 92.1-94.4%) for ER+/HER2- BC and 94.3% (95% CI: 93.1-95.5%) for ER+/HER2+ BC (p=0.08), while among patients with residual disease 5-year OS was 81.7% (95% CI: 81.1-82.2%) and 85.7% (95% CI: 84.5-86.9%) for the two subtypes respectively (p< 0.001). On multivariable analysis, omission of adjuvant ET was significantly associated with poorer OS in patients with residual disease for both ER+/HER2- BC (adjusted HR 1.72, p< 0.001) and ER+/HER2+ BC (adjusted HR 1.63, p< 0.001). In contrast, omission of adjuvant ET was not significantly associated with OS in patients with pCR, regardless of HER2 status (ER+/HER2- adjusted HR 1.28, p=0.20; ER+/HER2+ adjusted HR 1.13, p=0.54), Table 1. Conclusions: In pts receiving NAC for ER+ BC, those with ER+/HER2+ disease were less likely to have received adjuvant ET compared to ER+/HER2- patients, regardless of pCR. In pts with residual disease after NAC, omission of adjuvant ET was associated with significantly higher risk of death. These data provide strong support for interventions to increase utilization of ET, especially for patients with residual disease following NAC. The observation that ET omission did not impact OS in pts with ER+ BC who achieve pCR following NAC is hypothesis generating and may have implications for future de-escalation trials for this subset of patients. Table 1. Differential use of adjuvant endocrine therapy by subtype and pCR and the impact on overall survival Citation Format: Grace M. Choong, Judy C. Boughey, Tanya L. Hoskin, Courtney N. Day, Matthew P. Goetz. Impact of adjuvant endocrine therapy (ET) omission in ER+ breast cancer (BC) treated with neoadjuvant chemotherapy (NAC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-12.

Abstract Background: Data on the oncologic safety of omission of axillary lymph node dissection (ALND) in node positive (N+) patients who downstage to ypN0 with neoadjuvant chemotherapy (NAC) is sparse. Additionally, there is no consensus on which axillary staging procedure should be used in this setting, sentinel lymph node biopsy (SLNB) alone or in combination with localization and retrieval of the clipped positive node, also known as targeted axillary dissection (TAD). Whether the reduction in the false negative rate observed with TAD translates into a significant reduction in the rate of axillary recurrence is unknown. We sought to evaluate oncologic outcomes after omission of ALND in a large, real-world cohort of breast cancer (BC) patients and to compare rates of axillary recurrence after SLNB with dual tracer mapping vs. TAD. Methods: Data were collected from 19 centers in the Oncoplastic Breast Consortium (OPBC) and EUBREAST networks. Patients with T1-4 biopsy-proven N1-3 BC who underwent NAC followed by axillary staging with either SLNB with dual tracer mapping or TAD and who were pathologically node negative (ypN0) were included. ypN0 was defined as the absence of any tumor or isolated tumor cells. Competing risk analysis was performed to assess the cumulative incidence rates of axillary recurrence, locoregional recurrence, and any invasive (locoregional or distant) recurrence. Two-year cumulative incidence rates were compared between TAD and SLNB using the Gray’s test. Type I error rate was set to 0.05 (α). Results: We included 785 patients (565 treated with SLNB and 220 with TAD) treated with NAC followed by surgery from 01/2014-12/2020. Median patient age was 50 years. The majority (57%) of patients had clinical T2 tumors, and 95% had N1 disease. Most (55%) were HER2+, and 21% were triple negative. Most patients (81%) received anthracycline and taxane-based chemotherapy regimens, but NAC regimens differed between patients treated with TAD and those treated with SLNB (Table 1). All patients with HER2+ tumors received anti HER2 therapy. Nodal radiotherapy was administered to 76% of patients, and was more common in patients who underwent TAD (82% TAD vs 74% SLNB, p=0.017). Breast pathologic complete response (ypT0/is) was more frequent among those patients that had TAD (80% TAD vs. 66% SLNB, p< 0.001). TAD localization was with wire in 46%, radioactive seed in 40%, ultrasound in 5%, tattoo in 2%, and with a combination of these techniques in 7%. The clipped node was successfully retrieved in 94% of TAD cases. The median number of lymph nodes removed was lower in the TAD group compared to the SLNB group [3 (IQR 3-5) vs 4 IQR 3-5), p< 0.001], as was the median number of sentinel lymph nodes [3 (IQR 2-4) vs 4 IQR 3-5), p< 0.001] (Table 1). The 5-year rates of any axillary recurrence, locoregional recurrence, and any invasive recurrence in the entire cohort were 1.1% (95%CI 0.39-2.4%), 3.1% (95%CI 1.6-5.3%) and 10% (95%CI 7.6-13%), respectively. The two-year cumulative incidence of axillary recurrence did not differ between patients treated with TAD compared to SLNB (0% vs 0.9%, p=0.19). Conclusion: Early axillary recurrence after omission of ALND in patients who successfully downstage from N+ to ypN0 with NAC is a rare event following both SLNB or TAD, and was not significantly lower in TAD than SLNB. Although longer follow-up is needed to confirm these findings, the main advantage of TAD seems to be a reduction in the number of lymph nodes removed. Overall, these results support omission of ALND in patients who successfully downstage to node negative disease after NAC. Table 1: Clinicopathological Features of the Study Cohort, Stratified by Axillary Staging Technique Citation Format: Giacomo Montagna, Mary Mrdutt, Astrid Botty, Andrea V. Barrio, Varadan Sevilimedu, Judy C. Boughey, Tanya L. Hoskin, Laura H. Rosenberger, E Shelley Hwang, Abigail Ingham, Bärbel Papassotiropoulos, Bich Doan Nguyen-Sträuli, Christian Kurzeder, Danilo Diaz Aybar, Denise Vorburger, Dieter Michael Matlac, Edvin Ostapenko, Fabian Riedel, Florian Fitzal, Francesco Meani, Franziska Fick, Jacqueline Sagasser, Jörg Heil, Konstantin J. Dedes, Laszlo Romics, Maggie Banys-Paluchowski, Maria Del Rosario Cueva Perez, Marcelo Chavez Diaz, Martin Heidinger, Mathias K. Fehr, Mattea Reinisch, Nadia Maggi, Nicola Rocco, Nina Ditsch, Oreste Davide Gentilini, Regis Resende Paulinelli, Sebastian Sole Zarhi, Sherko Küemmel, Simona Bruzas, Simona Di Lascio, Tamara Parissenti, Uwe Güth, Valentina Ovalle, Christoph Tausch, Monica Morrow, Thorsten Kühn, Walter P. Weber. Oncological Outcomes Following Omission of Axillary Lymph Node Dissection in Node Positive Patients Downstaging To Node Negative with Neoadjuvant Chemotherapy: the OPBC-04/EUBREAST-06/OMA study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS4-02.

Abstract Purpose Breast cancer risk is elevated in pathogenic germline BRCA 1/2 mutation carriers due to compromised DNA quality control and elevated mutagenic load. We hypothesized that if immunosurveillance promotes tumor suppression, then normal breast lobules from BRCA carriers may demonstrate higher immune cell densities. Methods We assessed immune cell composition in normal breast lobules from age-matched women with progressively increased breast cancer risk, including 1) 19 women who donated normal breast tissue to the Komen Tissue Bank (KTB) at Indiana University Simon Cancer Center, 2) 15 women with biopsy-identified benign breast disease (BBD), and 3) 19 prophylactic mastectomies from women with germline mutations in BRCA1/2 genes. We performed immunohistochemical stains and analysis to quantitate immune cell densities from digital images in up to 10 representative lobules per sample. Median cell counts per mm2 were compared between groups using Wilcoxon rank-sum tests. Results Compared to BBD tissues, normal breast lobules from BRCA carriers had significantly higher densities of CD8 + cells (median 354.4 vs 200.0 cells/mm2, p = 0.01). Compared to KTB normal donor breast tissues, normal breast lobules from BRCA carriers also had significantly higher densities of CD4+ (median 116.3 vs 17.7 cells/mm2), CD8+ (354.4 vs 150.9 cells/mm2), CD11c+ (3.5% vs 0.4% pixels positive), and CD68+ (237.5 vs 57.8 cells/mm2) immune cells (each p < 0.001). Conclusions These preliminary analyses show that normal/benign breast lobules of BRCA mutation carriers contain increased immune cells compared with BBD and normal donated tissues. Future studies will be helpful to define relationships of mutations and immune responses.

AbstractBackgroundWe hypothesized full‐thickness chest wall resection (FTCWR) with advanced surgical techniques and modern systemic therapy is safe, provides local control, and good overall survival.MethodsRetrospective review of FTCWR (including rib or part of sternum) for breast cancer between 2000 and 2020. Primary endpoints included 90‐day morbidities and all‐cause mortality. Secondary endpoints were loco‐regional and distant recurrence, DFS and overall survival (OS).ResultsA total of 35 patients met the criteria. 34 FTCWR were for recurrence and the median time to chest wall recurrence was 6 years. Tumor subtype was triple‐negative in 51% and the remainder HR+ Her2−. 58% were palliative resections. FTCWR included rib(s) in 89% and portion of sternum in 57%; 94% required reconstruction and 80% were R0 resections. There were no 90‐day mortalities. Overall morbidity was 10/35(28%). 17(49%) patients received neoadjuvant systemic therapy for their recurrence and three received neoadjuvant radiation. Adjuvant treatment included chemotherapy (8), endocrine therapy (3), and both (8). Ten patients (28%) received adjuvant radiation. The Median follow‐up was 31 months and there were 6 (17%) loco‐regional and 7 (20%) distant recurrences. OS was 86% and 67% at 1 and 3 years, respectively.ConclusionFTCWR was associated with low morbidity, mortality, recurrence rates, and good OS. Selective FTCWR is safe and has acceptable short‐term survival rates.

Abstract Purpose: To evaluate prediction of response and event-free survival (EFS) following neoadjuvant endocrine therapy by SET2,3 index of nonproliferation gene expression related to estrogen and progesterone receptors adjusted for baseline prognosis. Experimental Design: A correlative study was conducted of SET2,3 measured from gene expression profiles of diagnostic tumor (Agilent microarrays) in 379 women with cStage II–III breast cancer from the American College of Surgeons Oncology Group Z1031 neoadjuvant aromatase inhibitor trial SET2,3 was dichotomized using the previously published cutoff. Fisher exact test was used to assess the association between SET2,3 and low proliferation at week 2–4 [Ki67 ≤ 10% or complete cell-cycle arrest (CCCA; Ki67 ≤ 2.7%)] and PEPI-0 rate in cohort B, and the association between SET2,3 and ypStage 0/I in all patients. Cox models were used to assess EFS with respect to SET2,3 excluding cohort B patients who switched to chemotherapy. Results: Patients with high SET2,3 had higher rate of pharmacodynamic response than patients with low SET2,3 (Ki67 ≤ 10% in 88.2% vs. 56.9%, P &lt; 0.0001; CCCA in 50.0% vs. 26.2%, P = 0.0054), but rate of ypStage 0/I (24.0% vs. 20.4%, P = 0.4580) or PEPI = 0 (28.4% vs. 20.6%, P = 0.3419) was not different. Patients with high SET2,3 had longer EFS than patients with low SET2,3 (HR, 0.52, 95% confidence interval: 0.34–0.80; P = 0.0026). Conclusions: This exploratory analysis of Z1031 data demonstrated a higher rate of pharmacodynamic suppression of proliferation and longer EFS in high SET2,3 disease relative to low SET2,3 disease. The ypStage 0/I rate and PEPI = 0 rate were similar with respect to SET2,3.

Abstract Introduction: The immune microenvironment of the breast is critical in the development and progression of breast cancer. By analyzing the immune components of breast tissues from patients at varying degrees of breast cancer risk, we may gain enhanced understanding of breast cancer risk and mechanisms of carcinogenesis. Methods: We compared the immune cell composition in breast lobules of age-matched patients with normal and benign breast disease (BBD) at progressively lower breast cancer risk: 1) 18 women with germline mutations in (BRCA1/2); 2) women at intermediate risk (BBD), and 3) women at normal risk (Komen Tissue Bank- (KTB)). We performed immunohistochemical stains for T lymphocytes (CD4, CD8); dendritic cells (CD11c); B lymphocytes (CD20) and macrophages (CD68) and quantitated immune cell densities in up to 10 representative lobules per sample. Results: The median age of all groups was 43.8 years (range: 34-67). BRCA carriers and BBD patients had a higher percentage of fibrocystic lobules (50% fibrocystic) compared to KTB samples (15% fibrocystic), p&lt;0.001. BRCA carriers had significantly higher densities of CD4, CD8, CD11c, and CD68 positive immune cells compared to KTB donor breast tissues, see Table. Compared with BBD patients, mutation carriers had higher densities of CD8+ cells and suggestively higher CD4+ cells, with similar results for other markers assessed. Conclusions: Preliminarily, these data showed that benign prophylactic breast tissues from BRCA mutation carriers contain increased numbers of several immune cell types versus tissues from patients at lower breast cancer risk, including BBD patients and research tissue donors. Further investigation is needed to confirm and expand these findings in relation to breast cancer risk. Table: Pairwise Wilcoxon rank-sum p-values comparing immune cell densities in breast lobules from women with BRCA germline mutations, vs BBD and KTB groups. All Lobules BRCA BBD KTB BRCA vs BBD BRCA vs KTB Median Median Median p-value p-value (IQR) (IQR) (IQR) CD4+, cells/mm2 116.3 63.8 17.7 0.08 &lt;0.001 (46.4-183.6) (25.0-121.1) (13.3-40.6) CD8+, cells/mm2 354.4 200 150.9 0.01 &lt;0.001 (285.5-473.2) (118.9-385) (110.1-225.0) CD11c+, % pp 3.5 3.4 0.4 0.66 &lt;0.001 (1.9-6.8) (1.6-4.7) (0-0.6) CD20+, cells/mm2 6.2 16.7 0 0.16 0.65 (0-25.4) (0-58.3) (0-20.8) CD68, cells/mm2 237.5 219 57.8 0.99 &lt;0.001 (122.9-281.8) (75.0-459.4) (29.2-100.0) IQR - Interquartile Range pp - pixel positive Citation Format: Joshua W. Ogony, Tanya L. Hoskin, Melody L. Stallings Mann, Stacey J. Winham, Rushin Brahmbhatt, Muhammad Asad Arshad, Alvaro Pena Jimenez, Teresa M. Allers, Mark E. Sherrman, Daniel W. Visscher, Derek C. Radisky, Amy C. Degnim. Immune biomarkers in breast tissues of patients with BRCA1/2 mutations, benign breast disease, and normal tissue donors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5947.

Abstract BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data.MethodsWe optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics.ResultsSamples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with CPM > 2). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.7 ng/ul and 1.68 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.58 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.803 in predicting QC status (pass/fail) of FFPE samples. ConclusionsWe provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.8 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis.