pCR In Triple-negative Breast Cancer Research Articles

Neoadjuvant Chemotherapy Response in Triple-Negative Apocrine Carcinoma: Comparing Apocrine Morphology, Androgen Receptor, and Immune Phenotypes.

Apocrine differentiation and androgen receptor (AR) positivity represent a specific subset of triple-negative breast cancer (TNBC) and are often considered potential prognostic or predictive factors. To evaluate the response of TNBC to neoadjuvant chemotherapy (NAC) and to assess the impact of apocrine morphology, AR status, Ki-67 labeling index (Ki-67LI), and tumor-infiltrating lymphocytes (TILs). A total of 232 TNBC patients who underwent NAC followed by surgical resection in a single institute were analyzed. The study evaluated apocrine morphology and AR and Ki-67LI expression via immunohistochemistry from pre-NAC biopsy samples. Additionally, pre-NAC intratumoral TILs and stromal TILs (sTILs) were quantified from biopsies using a deep learning model. The response to NAC after surgery was assessed based on residual cancer burden. Both apocrine morphology and high AR expression correlated with lower Ki-67LI (P < .001 for both). Apocrine morphology was associated with lower postoperative pathologic complete response (pCR) rates after NAC (P = .02), but the difference in TILs between TNBC cases with and without apocrine morphology was not statistically significant (P = .09 for sTILs). In contrast, AR expression did not significantly affect pCR (P = .13). Pre-NAC TILs strongly correlated with postoperative pCR in TNBCs without apocrine morphology (P < .001 for sTILs), whereas TNBC with apocrine morphology demonstrated an indeterminate trend (P = .82 for sTILs). Although TIL counts did not vary significantly based on apocrine morphology, apocrine morphology itself was a more reliable predictor of NAC response than AR expression. Consequently, although apocrine morphology is a rare subtype of TNBC, its identification is clinically important.

Prediction of pathologic complete response to chemoimmunotherapy in triple-negative breast cancer using tumor-infiltrating lymphocytes: Exploiting cutoff values.

e12629 Background: Triple-negative breast cancer (TNBC) prognosis is significantly influenced by tumor-infiltrating lymphocytes (TILs), but the lack of validated cutoff values has limited their clinical applicability. In the era of neoadjuvant pembrolizumab plus chemotherapy (P+CT) as a new standard of care, the role of TILs as predictors of response to chemoimmunotherapy remains uncertain. Methods: This study aimed to assess TILs as predictors of pathologic complete response (pCR) within the Neo-Real study, a multicenter, real-world data investigation on neoadjuvant P+CT in TNBC. TILs were evaluated using the standardized methodology of the International TILs Working Group. Logistic regression and receiver operating characteristic (ROC) curve analysis were performed to evaluate the predictive ability of TILs expression and multivariable models for pCR. Results: The analysis included 128 patients (pts) treated with neoadjuvant P+CT (68% stage II, 26% stage III). The proportion of pts in each TILs’ category and results of ROC curve analysis are presented in the Table. A cutoff value of 10% demonstrated the highest accuracy in predicting pCR, while a high specificity was observed at a cutoff value of 50%. Thus, the probability of residual disease if TILs ≥ 50% is considerably low. Incorporating categorized clinical and pathological variables, a multivariable logistic regression model, using TILs (≥ 10% vs &lt; 10%), Ki67 (≥ 50% vs &lt; 50%), and tumor stage (III vs II), exhibited the highest AUC (0.688) for predicting pCR. Conclusions: Our study underscores the predictive value of TILs for pCR in TNBC following neoadjuvant P+CT. The cutoff value of ≥ 50% identified patients with a very high probability of pCR. The results reinforce TILs’ use as a biomarker for treatment de-escalation, especially for pts with TILs ≥ 50%. Further enhancement of TILs' predictive potential may be achieved through multivariable models. [Table: see text]

Abstract PO4-03-07: The impact of chemotherapy relative dose intensity on pathological complete response in patients with triple negative breast cancer treated with neoadjuvant chemotherapy

Abstract Background: Neoadjuvant chemotherapy (NACT) is the preferred treatment approach for early-stage triple negative breast cancer (TNBC). Achieving pathologic complete response (pCR) after NACT is associated with improved event-free survival and overall survival in patients with TNBC. Major research efforts have been deployed to identify predictive biomarkers for pCR in TNBC. However, the values of chemotherapy (CT) relative dose intensity (RDI) in predicting pCR remain unclear. Methods: A retrospective analysis was conducted among 96 consecutive patients with early stage TNBC who received NACT between February 2018 and January 2023 at the University of Naples Federico II (Naples, Italy). Patients’ demographics, clinical-pathological features, and type of CT schedules were retrieved from electronic medical records. pCR was defined as the absence of residual invasive or in situ carcinoma in primary tumor and/or lymph nodes, upon chemotherapy administration. RDI was calculated as the ratio of delivered to planned CT dose intensity. RDI was defined as low if &amp;lt; 85% or high if ≥85%, respectively. Univariate and multivariate logistic regression were used to evaluate the association between age, tumor stage, RDI, CT regimen dose type, and pCR. Results: All patients were included in the analysis. Median age at diagnosis was 51 years (range 28-75). Sixty-five (68%) and 31 (32%) patients were diagnosed with stage II and stage III TNBC, respectively. Nineteen (19%) patients received a sequential schedule of anthracycline plus cyclophosphamide (AC) and a taxane (T). Sixty-one (64%) patients received AC followed by concurrent carboplatin-paclitaxel (CaP). Sixteen (17%) patients received the anti-PD1 pembrolizumab in addition to ACCaP chemotherapy backbone (ACCaP-Pem). Fifty-four (56%) patients received a dose-dense AC-based CT. A pCR was achieved in 59 (61%) patients. Interestingly, patients who received a dose-dense AC based neoadjuvant CT had higher chance to achieve a pCR compared with those treated with a non dose-dense AC based CT (70% vs 46%, p = 0.03). Overall, 79 (82%) and 17 (18%) patients received a RDI high and RDI low CT, respectively. No difference in terms of age, body mass index, performance status, tumor stage, type of chemotherapy was observed between the two groups (Table 1). At univariate analysis RDI high (OR 5.18, 95%CI: 1.65-16.31, p=0.005) and dose dense AC-based CT (OR 2.42, 95%CI: 0.18-0.97, p=0.04) were significantly associated with pCR. In a multivariate model including RDI, age, tumor stage, and AC regimen dose type, RDI was the only variable independently associated with pCR (OR 4.60, 95% CI: 1.40-15.06, p = 0.01). Conclusion: A chemotherapy RDI≥ 85% independently predicts pCR in early stage TNBC patients treated with standard NACT. In our cohort, one in five patients received a suboptimal chemotherapy dose intensity affecting at least in part tumor response. Studies with larger TNBC patient populations are warranted to confirm the impact of RDI on pCR and long-term outcomes. Table 1 Citation Format: Roberto Buonaiuto, Giuseppe Neola, Aldo Caltavituro, Paola Trasacco, Federica Pia Mangiacotti, Giovanna Pecoraro, Matteo Lambertini, Erica Pietroluongo, Pietro De Placido, Sabino De Placido, Valeria Forestieri, Mario Giuliano, Grazia Arpino, Carmine De Angelis. The impact of chemotherapy relative dose intensity on pathological complete response in patients with triple negative breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-07.

Identification of a Gene Panel Predictive of Triple-Negative Breast Cancer Response to Neoadjuvant Chemotherapy Employing Transcriptomic and Functional Validation.

Triple-negative breast cancer (TNBC) patients exhibiting pathological complete response (pCR) have better clinical outcomes compared to those with residual disease (RD). Therefore, robust biomarkers that can predict pCR may help with triage and resource prioritization in patients with TNBC. Herein, we identified a gene panel predictive of RD and pCR in TNBC from the discovery (n = 90) treatment-naive tumor transcriptomic data. Eight RD-derived genes were identified as TNBC-essential genes, which were highly predicative of overall survival (OS) and relapse-free survival (RFS) in an additional cohort of basal breast cancer (n = 442). Mechanistically, targeted depletion of the eight genes reduced the proliferation potential of TNBC cell models, while most remarkable effects were for combined SLC39A7, TIMM13, BANF1, and MVD knockdown in conjunction with doxorubicin. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and receiver operating characteristic curve (ROC) analyses revealed significant predictive power for the identified gene panels with an area under the curve (AUC) of 0.75 for the validation cohort (n = 50) to discriminate RD from pCR. Protein–Protein Interaction (PPI) network analysis of the pCR-derived gene signature identified an 87-immune gene signature highly predictive of pCR, which correlated with better OS, RFS, and distant-metastasis-free survival (DMFS) in an independent cohort of basal and, to a lesser extent, HER2+ breast cancer. Our data have identified gene signatures predicative of RD and pCR in TNBC with potential clinical implications.

Mid-treatment Ultrasound Descriptors as Qualitative Imaging Biomarkers of Pathologic Complete Response in Patients with Triple-Negative Breast Cancer.

This study aimed to investigate mid-treatment breast tumor ultrasound characteristics that may predict eventual pathologic complete response (pCR) in triple-negative breast cancer; specifically, we examined associations between pCR and two parameters: tumor response pattern and tumor appearance. Ultrasound was performed at mid-treatment, defined as the completion of four cycles of anthracycline-based chemotherapy and before receiving taxane-based chemotherapy. Consensus imaging review was performed while blinded to pathology results (i.e., pCR/non-pCR) from surgery. Tumor response pattern was described as "complete," "concentric," "fragmented," "stable" or "progression." Tumor appearance was designated as "mass," "architectural distortion," "flat tumor bed" or "clip only." Univariate and multivariate regression analyses of 144 participants showed significant associations between mid-treatment response pattern and pCR (p=0.0348 and p=0.0173, respectively), with complete and concentric response patterns more likely to achieve pCR than other patterns. Univariate and multivariate regression analyses further showed significant associations between mid-treatment tumor appearance and pCR (p < 0.0001 for both), with persistent appearance of mass less likely than other appearances to achieve pCR. To conclude, our study demonstrated strong associations between pCR and both tumor response pattern and tumor appearance, thereby suggesting that these parameters have potential as qualitative imaging biomarkers of pCR in triple-negative breast cancer.

Circulating Exosomal microRNAs as Predictive Biomarkers of Neoadjuvant Chemotherapy Response in Breast Cancer.

Background: Neoadjuvant chemotherapy (NACT) is an increasingly used approach for treatment of breast cancer. The pathological complete response (pCR) is considered a good predictor of disease-specific survival. This study investigated whether circulating exosomal microRNAs could predict pCR in breast cancer patients treated with NACT. Method: Plasma samples of 20 breast cancer patients treated with NACT were collected prior to and after the first cycle. RNA sequencing was used to determine microRNA profiling. The Cancer Genome Atlas (TCGA) was used to explore the expression patterns and survivability of the candidate miRNAs, and their potential targets based on the expression levels and copy number variation (CNV) data. Results: Three miRNAs before that NACT (miR-30b, miR-328 and miR-423) predicted pCR in all of the analyzed samples. Upregulation of miR-127 correlated with pCR in triple-negative breast cancer (TNBC). After the first NACT dose, pCR was predicted by exo-miR-141, while miR-34a, exo-miR182, and exo-miR-183 predicted non-pCR. A significant correlation between the candidate miRNAs and the overall survival, subtype, and metastasis in breast cancer, suggesting their potential role as predictive biomarkers of pCR. Conclusions: If the miRNAs identified in this study are validated in a large cohort of patients, they might serve as predictive non-invasive liquid biopsy biomarkers for monitoring pCR to NACT in breast cancer.

Abstract PD6-07: Volumetric changes on longitudinal dynamic contrast enhanced MR imaging (DCE-MRI) as an early treatment response predictor to neoadjuvant systemic therapy (NAST) in triple negative breast cancer (TNBC) patients

Abstract Background and Purpose:There is currently a lack of recognized imaging criteria for prediction of treatment response to NAST in breast cancer patients with recent reports showing that breast MRI is the most accurate modality for evaluation of NAST response. DCE-MRI evaluates tumor perfusion that influences tumor enhancement at the post-contrast subtraction images and allows for more accurate measurement of changes in tumor volume during NAST. In this study, we evaluated the ability of tumor volumetric changes after 2 and 4 cycles of NAST by longitudinal ultrafast DCE-MRI to predict pathologic complete response (pCR) in TNBC undergoing NAST. Materials and Methods: Stage I-III TNBC patients enrolled in an IRB approved prospective clinical trial (ARTEMIS, NCT02276433) who had ultrafast DCE-MRI at baseline (BL, N=103), post 2 cycles (C2, N=59), and post 4 cycles (C4, N=103) of anthracycline-based NAST,and had surgery, were included in this analysis. Tumor volume was calculated using 3D measurements of the index lesion at BL, C2, and C4. Percent change of tumor volume (%TV) between BL, C2, and C4 was calculated at early (9-12 sec) and delayed (360-480 sec) phases of DCE-MRI. The largest lesion was used for analysis in patients with multicentric or multifocal disease. Demographic, clinical, and pathologic data and treatment response at surgery (pCR versus non-pCR) were documented. Receiver operating characteristics curve (ROC) analysis was performed for prediction of pCR status. Positive predictive value (PPV), negative predictive value (NPV) and Youden Index were used to select %TV cut-off thresholds for pCR prediction.Results: 103 patients (median age, 53 years; range, 24-79 years) were included, 48 (47%) had pCR, and 55 (53%) had non-pCR at surgical pathology. The %TV reduction at C2 DCE-MRI was predictive of pCR on both early phase DCE MRI (AUC, 0.873; CI:0.779-0.968, p &amp;lt; .0001) and delayed phase DCE MRI (AUC, 0.844; CI:0.742-0.947, p &amp;lt; .0001). Optimal thresholds were as follows: 70% TV reduction on early phase DCE MRI with Youden’s index of 1.58 was able to predict pCR correctly for 79% of patients with PPV of 81%; 75% TV reduction on delayed phase with Youden’s Index of 1.44 was able to predict pCR correctly for 71% of patients with PPV of 85%.%TV reduction was also predictive of pCR at the C4 time point on both early phase DCE MRI (AUC, 0.761; CI:0.665-0.856, p &amp;lt; .0001) and delayed phase DCE MRI (AUC, 0.737; CI:0.641-0.833, p &amp;lt; .0001). Optimal thresholds were as follows: 90% TV reduction on early phase DCE MRI with Youden’s index of 1.43 was able to correctly predict pCR in 72% of patients with PPV of 70%; and 90% TV reduction on delayed phase with Youden’s Index of 1.34 was able to predict pCR correctly in 68% of patients with PPV of 71%.Conclusion: Our data shows that percent tumor volume reduction by DCE-MRI after 2 and 4 cycles of NAST was able to predict pCR in TNBC with high accuracy and can be used as an early imaging biomarker of NAST response prediction. Volumetric changes by longitudinal DCE-MRI can be used to differentiate chemoresistant and chemosensitive TNBC patients as early as after 2 cycles of NAST, and can help to triage patients for treatment de-escalation or targeted therapy. Citation Format: Gaiane Margishvili Rauch, Adrada E Beatriz, Rosalind P Candelaria, Nabil Elshafeey, Abeer H Abdelhafez, Benjamin C Musall, Jia Sun, Medina Boge, Rania M.M Mohamed, Jong Bum Son, Shu Zhang, Jessica Leung, Deanna Lane, Marion Scoggins, David Spak, Elsa Arribas, Lumarie Santiago, Gary J Whitman, Huong T. Le-Petross, Tanya W Moseley, Jason B. White, Elizabeth Ravenberg, Ken-Pin Hwang, Peng Wei, Lei Huo, Jennifer K Litton, Vicente Valero, Debu Tripathy, Alastair M Thompson, Mark D Pagel, Jingfei Ma, Wei T Yang, Stacy Moulder. Volumetric changes on longitudinal dynamic contrast enhanced MR imaging (DCE-MRI) as an early treatment response predictor to neoadjuvant systemic therapy (NAST) in triple negative breast cancer (TNBC) patients [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 PD6-07.

Predictive markers for pathological complete response after neo-adjuvant chemotherapy in triple-negative breast cancer

A combination of Sox10 and GATA3 was previously identified as a marker for metastatic triple-negative breast cancer (TNBC), but it is uncertain whether their expression is associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). This study investigates the predictive value of clinicopathological characteristics, as well as protein expression of Sox10, GATA3, p53 and p63, in a consecutive series of TNBC patients treated with NAC.Archived hematoxylin & eosin stained slides of core biopsies and resection specimens from 35 TNBC patients were reviewed. The following clinicopathological characteristics were determined at the biopsy level: age at diagnosis, cancer type, Nottingham grade, lympho-vascular invasion, syncytial growth, necrosis, clear cell differentiation, myxoid peritumor stroma, stromal tumor-infiltrating lymphocytes (sTILs) and presence of an in situ component. The MD Anderson residual cancer burden (RCB) score and corresponding RCB class were determined. Immunohistochemistry for Sox10, p53, GATA3 and p63 was performed at the biopsy level.sTILs, either as a continuous or as a dichotomous variable, were the only parameter that was significantly associated with pCR in univariable and multivariable analyses. Assessment of sTILs showed moderate to good interobserver agreement. High sTILs (≥40%) were significantly associated with increased pCR rates, and this association was observer-independent.This retrospective study of a consecutive community-based cohort of TNBC patients confirms that sTILs are a robust, observer-independent predictor for therapeutic response after NAC. The combination of Sox10, GATA3 and p53 immunoreactivity is unlikely to harbor any predictive value for pCR in TNBC.

Neoadjuvant Treatment for Triple Negative Breast Cancer: Recent Progresses and Challenges.

Triple negative breast cancer (TNBC) is an aggressive breast cancer with historically poor outcomes, primarily due to the lack of effective targeted therapies. The tumor molecular heterogeneity of TNBC has been well recognized, yet molecular subtype driven therapy remains lacking. While neoadjuvant anthracycline and taxane-based chemotherapy remains the standard of care for early stage TNBC, the optimal chemotherapy regimen is debatable. The addition of carboplatin to anthracycline, cyclophosphamide, and taxane (ACT) regimen is associated with improved complete pathologic response (pCR). Immune checkpoint inhibitor (ICI) combinations significantly increase pCR in TNBC. Increased tumor infiltrating lymphocyte (TILs) or the presence of DNA repair deficiency (DRD) mutation is associated with increased pCR. Other targets, such as poly-ADP-ribosyl polymerase inhibitors (PARPi) and Phosphatidylinositol-3-kinase/Protein Kinase B/mammalian target of rapamycin (PI3K-AKT-mTOR) pathway inhibitors, are being evaluated in the neoadjuvant setting. This review examines recent progress in neoadjuvant therapy of TNBC, including platinum, ICI, PARPi, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) pathway targeted therapies, and novel tumor microenvironment (TME) targeted therapy, in addition to biomarkers for the prediction of pCR.

Abstract P1-02-04: Contrast-enhanced ultrasonography after two cycles of chemotherapy predicts the outcome of neoadjuvant chemotherapy in triple-negative breast cancer

Abstract Background: Contrast-enhanced ultrasonography (CEUS) allows a thorough visualization of the intra-tumoral blood flow. This study aimed to determine if CEUS can predict the early effects of neoadjuvant chemotherapy (NAC) on breast cancer. Patients and Methods: We evaluated 93 patients with triple-negative type (ER-negative and HER2-negative, n= 30), HER2-positive (HER2-positive and any ER, n= 34), and luminal type (ER-positive and HER2-negative, n= 29) invasive breast cancer who underwent NAC between September 2012 and March 2019. Ultrasonography (US) and CEUS were performed before NAC and after two cycles of NAC. All patients underwent either mastectomy or breast-conserving surgery after NAC. To objectively evaluate CEUS findings, the ascending slope (AS) in perfusion parameters of CEUS was calculated using time-intensity curves based on enhancement intensity and temporal changes. The changes of maximum tumor diameter in US (ΔUS) and the changes of AS in perfusion parameters of CEUS (ΔAS) were measured in the primary tumor. Results: Thirty-seven (39.8%) patients achieved pathological complete response (pCR). In triple-negative breast cancer, both of ΔUS and ΔAS showed a significant decline with pCR, but there were no significant differences in ΔUS and ΔAS in the luminal-type and HER2-positive breast cancer. We set cutoffs of the ΔUS (-37%, AUC 0.742) and ΔAS (-26%, AUC 0.823) for predicting pCR on the basis of their ROC curves. ΔUS (OR 8.75, p=0.017) and ΔAS (OR 21.3, p=0.0001) in univariate analysis and ΔAS (OR 16.1, p=0.009) in multivariate analysis were significant predictors for pCR in triple-negative breast cancer. The sensitivity, specificity, and accuracy for predicting pCR in triple-negative breast cancer were 0.70, 0.79, and 0.76 in ΔUS and 0.80, 0.84, and 0.83 in ΔAS, respectively. The sensitivity, specificity, and accuracy of ΔAS were greater than those of ΔUS for predicting pCR. Conclusions: CEUS may serve as a new important diagnostic modality when planning therapeutic strategies, for patients with triple-negative breast cancer who received NAC. univariate analysis and multivariate analysis for pCR in triple-negative breast cancerCharacteristicUnivariate logistic analysisMultivariate logistic analysisOR95%CIpOR95%CIpAge, &amp;lt;50 vs ≧500.960.21-4.341.0000Clinical T, T1 vs T2-40.900.13-6.461.0000Clinical N, Negative vs Positive0.360.08-1.720.2635Nuclear Grade, 3 vs 1-20.750.14-3.901.0000Ki67, &amp;lt;20 vs ≧ 20--0.2685US findingsΔUS(%), ≧-37 vs ←378.751.53-50.110.01695.800.73-58.390.1004ΔAS(%), ≧-26 vs ←2621.332.94-154.550.001316.142.28-177.580.0096 Citation Format: Mai Nishina, Norio Masumoto, Akiko Kanou, Kayo Fukui, Yuri Kimura, Ai Amioka, Tomoko Itagaki, Shinsuke Sasada, Akiko Emi, Takayuki Kadoya, Morihito Okada. Contrast-enhanced ultrasonography after two cycles of chemotherapy predicts the outcome of neoadjuvant chemotherapy in triple-negative breast cancer [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 P1-02-04.

The role of stromal tumor infiltrating lymphocytes (sTILs) as a predictive biomarker for carboplatin-based neoadjuvant chemotherapy (NACT) in triple-negative breast cancer (TNBC).

e12085 Background: High numbers of sTILs are predictive of pCR in TNBCs. This study examines the role of sTILs as a predictive biomarker for carboplatin (Cb) based NACT. Methods: sTILs were scored on 88 pre NACT TNBC biopsies as per the International TILs Working Group and were scored as continuous and categorical variables: 0-10%; 11-25%; 26-49%; ≥50%. OR was calculated using non-pCR as baseline parameter. Results: Patients with sTILs &gt; 10% had increased pCR rates compared to those with sTILs ≤10%: pCR breast (B) (61% vs 31% p = 0.004); pCR breast/axilla (BA) (52% vs 29% p = 0.021). In those treated with Cb, there were no differences in pCR B between sTILs &gt; 10% or ≤10% (67% vs 53%; p = 0.355) or pCR BA (60% vs 53%; p = 0.500). In those who did not receive Cb, sTILs &gt; 10% had increased pCR B (57% vs 15%; p = 0.002) and pCR BA (46% vs 12%; p = 0.005) compared to sTILs ≤10%. Similar trends were seen with sTILs &gt; 25% and ≥50%: differences were seen in patients who did not receive Cb, but not in those treated with Cb. In LPBCs, the addition of Cb did not significantly increase pCR rates: 67% vs 83% for Cb and non-Cb regimens (p = 0.583). In non-LPBC, pCR rates were increased with Cb: pCR B 59% vs 31% (p = 0.017), and pCR BA 56% vs 23% (p = 0.005) for Cb and non-Cb regimens. The association between sTILs and pCR was significant in multivariable models adjusting for grade and Cb. In patients who did not receive Cb (n = 54), increasing sTILs were associated with increased pCR B (OR 0.15 p = 0.004; OR 0.27 p = 0.039; OR 0.10 p = 0.05 for &gt; 10%, &gt; 25%, ≥50%) and pCR BA (OR 0.18 p = 0.019; OR 0.08 p = 0.029 for &gt; 10%, ≥50%). In patients treated with Cb (n = 30), increasing sTILs were not associated with pCR B (OR 1.97 p = 0.581; OR 0.27 p = 0.334; OR 0.50 p = 0.719) or pCR BA (OR 2.93 p = 0.372; OR 0.79 p = 0.831; OR 0.40 p = 0.622 for &gt; 10%, &gt; 25%, ≥50%). Conclusions: sTILs are predictive of pCR: as sTILs increase, pCR rates increase. Tumors with high sTILs had high pCR rates to anthracycline-taxane (AT) NACT. The effect of increasing sTILs on pCR was most notable in patients who did not receive Cb based NACT, suggesting that tumors with high sTILs are inherently sensitive to AT based chemotherapy. Intensifying NACT with Cb could be used to increase pCR rates in patients with low sTILs. sTILs should be explored as a biomarker to intensify chemotherapy in those with low sTILs, and to de-escalate chemotherapy in tumors with high sTILs.

A two-gene epigenetic signature for the prediction of response to neoadjuvant chemotherapy in triple-negative breast cancer patients

BackgroundPathological complete response (pCR) after neoadjuvant chemotherapy (NAC) in triple-negative breast cancer (TNBC) varies between 30 and 40% approximately. To provide further insight into the prediction of pCR, we evaluated the role of an epigenetic methylation-based signature.MethodsEpigenetic assessment of DNA extracted from biopsy archived samples previous to NAC from TNBC patients was performed. Patients included were categorized according to previous response to NAC in responder (pCR or residual cancer burden, RCB = 0) or non-responder (non-pCR or RCB > 0) patients. A methyloma study was performed in a discovery cohort by the Infinium HumanMethylation450 BeadChip (450K array) from Illumina. The epigenetic silencing of those methylated genes in the discovery cohort were validated by bisulfite pyrosequencing (PyroMark Q96 System version 2.0.6, Qiagen) and qRT-PCR in an independent cohort of TN patients and in TN cell lines.ResultsTwenty-four and 30 patients were included in the discovery and validation cohorts, respectively. In the discovery cohort, nine genes were differentially methylated: six presented higher methylation in non-responder patients (LOC641519, LEF1, HOXA5, EVC2, TLX3, CDKL2) and three greater methylation in responder patients (FERD3L, CHL1, and TRIP10). After validation, a two-gene (FER3L and TRIP10) epigenetic score predicted RCB = 0 with an area under the ROC curve (AUC) = 0.905 (95% CI = 0.805–1.000). Patients with a positive epigenetic two-gene score showed 78.6% RCB = 0 versus only 10.7% RCB = 0 if signature were negative.ConclusionsThese results suggest that pCR in TNBC could be accurately predicted with an epigenetic signature of FERD3L and TRIP10 genes. Further prospective validation of these findings is warranted.

Abstract P1-15-07: Phase II trial of neoadjuvant carboplatin and nab-paclitaxel in patients with locally advanced triple negative breast cancer

Abstract Background: Response to neoadjuvant therapy (NT) predicts progression-free and overall survival in triple negative breast cancer (TNBC). Carboplatin has shown efficacy in patients with TNBC. The current phase II prospective neoadjuvant trial was designed to decrease toxicities and improve efficacy. Methods: Patients with TNBC received carboplatin (carb) and nab-paclitaxel (nab). Pre-NT biopsies were procured to evaluate for biological predictors of pathological complete response (pCR). Newly diagnosed stage II-III patients with TNBC were treated with 4 cycles of carb (AUC 6, day 1 of 28 day cycle) and weekly nab 100 mg/m2 x 16. Targeted accrual goal is 70. RNA extracted from formalin fixed paraffin embedded (FFPE) biopsies pre-NT was tested for MammaPrint/BluePrint and custom Agilent full genome microarrays for gene expression (GE, by Agendia Inc). The raw gMeanSignal was log2 transformed and normalized to the 75thpercentile for GE analysis. Association between MammaPrint/ BluePrint results and pCR was tested by Fisher exact test. The linear model from R limma package was applied. Ingenuity Pathway Analysis (IPA) was applied to assess functional pathways associated with pCR. Cellular distribution by CIBERSORT analysis was carried out to estimate the abundance of 22 different cell types in each patient sample, and test whether the distribution of cell types is different between pCR and non-responders. Results: A total of 64 patients were enrolled. Two patients were deemed ineligible (Her2+), and three were too early, resulting in 59 patients evaluable for pathological response. The pCR rate was 47% (RCB0, 28/59). Eight patients had RCB I. RCB0 plus RCBI reached 61%. Sufficient quality RNA and DNA were available from the first 43 of 55 pts with TNBC. 44/59 (75%) required dose modifications (mostly hematologic), 5 patients had grade 3 peripheral neuropathy (PN), 3 had grade 2 PN, and 3 patients had grade 2 LFTs. In the 53 pts with GE assessment, pCR was inversely associated with luminal BluePrint type (p=0.04). With fold change &amp;gt;1.5 and p-value &amp;lt; 0.05, 36 genes were differentially expressed (DE) in TNBC. CIBERSORT analysis suggested that T-cell regulatory cells (TREGS) were associated with pCR in TNBC, and 5 cell types (plasma cells, TREGS, macrophage, dendritic cells and neutrophils) presented differently between all pCR and non-pCRs with P-value &amp;lt;0.05. TDP analysis to assess correlation with pCR is ongoing. Conclusions: The combination of carboplatin and nab-paclitaxel given in the neoadjuvant setting reached a promising pCR rate of 47%. The MammaPrint non-luminal BluePrint subtype was predictive of pCR in TNBC. Preliminary analysis suggested that a 36-gene signature for TNBC was associated with pCR. CIBERSORT analysis revealed 5 cell types with different abundance between the pCR and non-responders, suggesting the need to target the tumor microenvironment. Citation Format: Yuan Y, Frankel P, Li M, Kruper L, Jones V, Treece T, Waisman J, Yim J, Tumyan L, Schmolze D, Hurria A, Yeon C, Mortimer J, Somlo G. Phase II trial of neoadjuvant carboplatin and nab-paclitaxel in patients with locally advanced triple negative breast cancer [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 P1-15-07.

TILGen: A Program to Investigate Immune Targets in Breast Cancer Patients - First Results on the Influence of Tumor-Infiltrating Lymphocytes

Background: Despite advancements in the treatment of primary and metastatic breast cancer, many patients lack a durable response to these treatments. Patients with triple-negative breast cancer (TNBC) and human epidermal growth factor receptor 2(HER2)-positive breast cancer who do not have a pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) have a very poor prognosis. Tumor-infiltrating lymphocytes (TILs) have been identified as a predictive marker for pCR after NACT in TNBC and HER2-positive breast cancer. These patient populations could also be suitable for novel treatment strategies including neoepitope-based therapies. This work analyses the effect of TILs on the pCR in neoadjuvantly treated patients in the TILGen study and presents the procedures aimed at establishing neoepitope-based therapies in this study. Methods: Neoadjuvantly treated HER2-positive and TNBC patients were eligible for the presented analysis concerning the association between TILs and pCR. A total of 146 patients could be identified within the TILGen study. TILs were evaluated as percentage of stromal tumor tissue in core biopsies at primary diagnosis. The phenotype ‘lymphocyte-predominant breast cancer' (LPBC) was associated with pCR by logistic regression adjusted for estrogen receptor status, progesterone receptor status, HER2 status, age at diagnosis, and grading. Results: LPBC was seen in 24 (16.4%) patients. In this patient group, 66.7% achieved a pCR, while the pCR rate was 32.8% in patients with a low TIL count. The adjusted odds ratio was 6.60 (95% confidence interval 2.02-21.56; p < 0.01). Conclusion: TILs are a strong predictor of pCR in TNBC and HER2-positive breast cancer patients. Implications for the use of this information including the effect on prognosis might help to identify patients most likely to benefit from a neoepitope-based therapy approach.

Abstract PD1-03: Multivariate analysis of subtype and gene expression signatures predictive of pathologic complete response (pCR) in triple-negative breast cancer (TNBC): CALGB 40603 (Alliance)

Abstract Support: U10CA180821, U10CA180882 Background: The addition of either carboplatin (Cb) or bevacizumab (Bev) to standard neoadjuvant chemotherapy (NACT) increases pCR rates in TNBC overall and in the dominant subset of basal-like cancers (Sikov et al, JCO 2015; Sikov et al, SABCS 2014). Multigene expression signatures more accurately reflect tumor biology for response prediction and prognosis than individual gene expression. We evaluated the ability of multivariate analysis of gene expression signatures to create predictive models for achievement of pCR in TNBC. Methods: RNA sequencing was successful on 389 pretreatment samples from patients with available pCR data, and used to assign PAM50 subtype and calculate gene signatures scores for 489 published expression signatures. Elastic net, a penalized regression model for high dimensional variable selection, was used to select features associated with pCR in all TNBC and in the basal-like subset. Models were derived in a training set (2/3 of samples) and validated in a separate test set (1/3). A separate model was derived using 196 TNBC samples from patients treated only on the standard NACT +/- Cb arms for application to external TNBC neoadjuvant data sets not treated with Bev. Results: Consistent with our prior partial data set, 343 (88%) of the cancers were classified basal-like, in whom the in breast pCR rate was 54%; the remainder were classified normal-like (n=32) or HER2-enriched (n=14) with a non-basal pCR rate of 56%. Elastic Net analysis in all TNBC generated a model of 23 signatures and treatment assignment with 68% sensitivity and 64% specificity. The area under the curve was 0.64 (p-value=0.0019). Nineteen modules, including immune cell signatures (Th1, NK, IgG), immunoglobulin variable region expression, addition of Cb and Bev and expression of genes at regions 15q25, 17p11.2-13.3, and 8p22 were positively associated with response. The latter two regions are associated with aggressive breast cancer, and while not part of the 17p13 signature, this region contains TP53, a gene important in TNBC. Six modules were associated with resistance, including luminal progenitor, TGFB, NOTCH, FOS/JUN, 8p amplicon, and eosinophil signatures. When limited to basal-like samples, a model including 32 modules and addition of Cb and Bev was generated, with 62.3% sensitivity and 59.1% specificity. Seventeen features were selected in both models. Omitting Bev-treated patients, a model using just the gene expression signatures was developed. The predictive value of this model will be assessed using an external cohort of TNBC patients treated with neoadjuvant docetaxel and Cb (NCT01560663) and results presented. Conclusions: Multivariate analysis of gene expression signatures derived from pretreatment samples enabled the construction of models to predict achievement of pCR in TNBC. These models performed well on our test set, and will be assessed for their predictive ability in other TNBC data sets. If validated by future analyses, this could help us identify patients likely to achieve pCR with standard NACT and may benefit from the addition of agents such as Cb or Bev. ClinicalTrials.gov Identifier: NCT00861705. Citation Format: Hoadley KA, Hyslop T, Fan C, Berry DA, Hahn O, Tolaney SM, Sikov WM, Perou CM, Carey LA. Multivariate analysis of subtype and gene expression signatures predictive of pathologic complete response (pCR) in triple-negative breast cancer (TNBC): CALGB 40603 (Alliance) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD1-03.

Association between distinct intratumoral and stromal tumor infiltrating immune cells and pathologic complete response (pCR) in different breast cancer subtypes after neoadjuvant chemotherapy (NAC).

1033 Background: Tumor-infiltrating lymphocytes (TILs) are emerging as prognostic factors in early breast cancer and have shown to predict response to NAC, especially in triple-negative breast cancer (TNBC). However, it is not clear whether all intratumoral and stromal immune cells show the same predictive value or if there is a specific cell subtype involved in response and prognosis Methods: We retrospectively analyzed the diagnostic biopsies of breast cancer patients treated with NAC in our institution in the last 4 years. 3-4µm thick sections were subjected to immunohistochemistry using antibodies against CD3, CD4, CD8 and CD20. Intratumoral and stromal lymphocytes were measured and expressed in positive cells per mm². Every patient received chemotherapy with weekly paclitaxel and doxorubicin-cyclophosphamide and HER2 positive tumors were treated with trastuzumab. All patients received surgery. pCR was defined as no residual invasive tumor in the breast and the axilla. Descriptive statistics are reported as median (IQR, interquartile range) or mean (SD, standard deviation) for continuous variables, and as absolute or relative frequencies for categorical variables Results: From 144 evaluable specimens, 72 were estrogen-receptor and/or progesterone-receptor positive, 27 were TNBC and 45 were HER2 positive tumors. 70 pCR were documented. The expression of stromal CD3, CD4 and CD20 lymphocytes was significantly associated with pCR in all breast cancer subtypes: CD3 (28,00 [27,25], p < 0.001); CD4 (16,00 [23,75], p = 0.007); CD20 (2,50 [7,25], p = 0.046). In the subgroup analysis, both stromal CD3 (34,00 [24,00], p = 0.004), intratumoral CD3 (7,50 [19,50], p = 0.013), and intratumoral CD8 (3,00 [17,50], p = 0.041) lymphocytes were significantly associated with pCR in TNBC. In HER2 positive tumors stromal CD3 and CD4 were predictive of pCR ((26,00 [26,00], p < 0.001) and (16,00 [24,00], p = 0.042) respectively) Conclusions: Stromal CD3 lymphocytes are the most significant predictive factor of pCR in all breast cancer subtypes after NAC. In TNBC both intratumoral and stromal CD3 expression was associated with pCR.

Abstract P3-07-56: Predictive value of tertiary lymphoid structure assessed by high endothelial venule count in neoadjuvant setting of triple-negative breast cancer

Abstract Background Tertiary lymphoid structure (TLS) is an ectopic lymph node-like structure characterized by lymphoid aggregation with high endothelial venules (HEVs), and is an important source of tumor-infiltrating lymphocytes (TILs). TILs have a strong prognostic and predictive significance, particularly in triple-negative breast cancer (TNBC). We previously analyzed expression of immune-related genes in pre-neoadjuvant chemotherapy (NAC) biopsy samples using NanoString assay and showed that gene expression of follicular helper T cell marker CXCL13, which is closely associated with TLSs, was an independent predictive factor for pathologic complete remission (pCR) in TNBC. However, measuring gene expression of biopsy sample is not easy to perform in daily pathology practice. Therefore, we evaluated TLSs by assessing hematoxylin and eosin (H&amp;E) stained slides and immunohistochemistry, and investigated their clinical importance. Methods A total of 108 patients diagnosed with primary TNBC and treated with NAC (anthracycline, cyclophosphamide, and taxane) were included. The amounts of TILs and TLSs were histopathologically measured in H&amp;E slides. Immunohistochemical studies were done in 55 cases with available tissue samples. The numbers of CD3-, CD8-, and CD20- immunopositive cells in tumor areas were counted by the image analyzer. MECA79- immunopositive HEV densities were calculated. Their relationship to CXCL13 gene expression by NanoString assay was also analyzed. Results The overall rate of pCR was 30.6% (33 of 108 tumors). Lower pre-NAC clinical T stage and higher level of TIL and TLS assessed by H&amp;E slides were predictors of pCR in all cases. The mean number of MECA 79-immunopositive HEV in pre-NAC biopsy samples was 12 (range, 0-72). The amounts of TILs and TLSs, numbers of CD3-, CD8-, and CD20- positive cells, HEV density, and expression of CXCL13 showed good correlation with one another. Higher HEV density, CD20- positive cell number, and CXCL13 expression were predictors of pCR. Higher CD8- positive cell numbers and CXCL13 expression were associated with better disease-free survival rate. Conclusion The amount of TLSs assessed by H&amp;E slides and MECA 79-immunopositive HEV densities was well correlated with level of TILs, numbers of CD3-, CD8-, and CD20-positive cells, and gene expression of CXCL13, and was significantly associated with pCR in TNBCs. Therefore, assessing HEV density by MECA 79 immunohistochemistry in pre-NAC biopsy samples might be an objective and valuable tool for predicting pCR of TNBC in routine pathology practice. Further investigation of mechanism of TLS development might help to improve immunotherapeutic strategy. Citation Format: Song IH, Lee HJ, Park IA, Yu JH, Ahn J-H, Gong G. Predictive value of tertiary lymphoid structure assessed by high endothelial venule count in neoadjuvant setting of triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-56.

Predictive significance of DNA damage and repair biomarkers in triple-negative breast cancer patients treated with neoadjuvant chemotherapy: An exploratory analysis.

Response of cancer cells to chemotherapy-induced DNA damage is regulated by the ATM-Chk2 and ATR-Chk1 pathways. We investigated the association between phosphorylated H2AX (γ-H2AX), a marker of DNA double-strand breaks that trigger the ATM-Chk2 cascade, and phosphorylated Chk1 (pChk1), with pathological complete response (pCR) in triple-negative breast cancer (TNBC) patients treated with neoadjuvant chemotherapy. γ-H2AX and pChk1 were retrospectively assessed by immunohistochemistry in a series of pretreatment biopsies related to 66 patients. In fifty-three tumors hormone receptor status was negative in both the diagnostic biopsies and residual cancers, whereas in 13 cases there was a slight hormone receptor expression that changed after chemotherapy. Internal validation was carried out. In the entire cohort elevated levels of γ-H2AX, but not pChk1, were associated with reduced pCR rate (p = 0.009). The association tested significant in both uni- and multivariate logistic regression models (OR 4.51, 95% CI: 1.39-14.66, p = 0.012, and OR 5.07, 95% CI: 1.28-20.09, p = 0.021, respectively). Internal validation supported the predictive value of the model. The predictive ability of γ-H2AX was further confirmed in the multivariate model after exclusion of tumors that underwent changes in hormone receptor status during chemotherapy (OR 7.07, 95% CI: 1.39-36.02, p = 0.018). Finally, in residual diseases a significant decrease of γ-H2AX levels was observed (p < 0.001). Overall, γ-H2AX showed ability to predict pCR in TNBC and deserves larger, prospective studies.

Impact of weight change during neoadjuvant chemotherapy on pathologic response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an uncommon but aggressive subtype of breast cancer. Obesity has been associated with an increased risk of breast cancer and worse prognosis. Some studies suggest that obese patients are less likely to achieve pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) and experience worse overall survival. Ki-67 is a proliferation marker that correlates with tumor aggressiveness. The goal of this study was to examine the impact of weight change during NCT for TNBC on pathologic response and Ki-67 reduction. Retrospective review identified 173 TNBC patients treated between 2004 and 2011. Data were collected on patient demographics, pre- and post-NCT body mass index (BMI), Ki-67, and pCR. Data analysis was performed using the two-tailed Student's t-test, analysis of variance (ANOVA), and Fisher's exact test. Sixty-six patients met final study criteria. Forty-three patients lost weight during chemotherapy and 23 gained weight. Patients in the weight gain group were significantly younger (P = 0.0013). There was no significant difference between the two groups in terms of Ki-67 reduction (P = 0.98) or pCR (P = 0.58). When patients were separated into normal weight (BMI<25 kg/m2), overweight (BMI ≥ 25 and <30 kg/m2), and obese (BMI ≥ 30 kg/m2), there was no significant difference in Ki-67 among those groups either before or after NCT. The degree of obesity did not have a significant impact on Ki-67 reduction. Weight change during NCT does not appear to correlate with Ki-67 change or achieving pCR in TNBC. This may reflect the nature of this subtype of breast cancer that is less responsive to the hormonal effects that adipose tissue exerts on cancer cell proliferation.

Pre-surgery Carboplatin and Bevacizumab Increase pCR in Triple-Negative Breast Cancer

Pre-surgery Carboplatin and Bevacizumab Increase pCR in Triple-Negative Breast Cancer