Background The generation of data capturing the risk-benefit ratio of incorporating carboplatin (Cb) to neoadjuvant chemotherapy (NACT) for triple-negative breast cancer (TNBC) in a clinical practice setting is urgently needed. Tumour-infiltrating lymphocytes (TILs) have an established role in TNBC receiving NACT, however, the role of TIL dynamics under NACT exposure in patients receiving the current standard of care is largely uncharted. Methods Consecutive TNBC patients receiving anthracycline-taxane [A-T] +/− Cb NACT at three Institutions were enrolled. Stromal-TILs were evaluated on pre-NACT and residual disease (RD) specimens. In the clinical cohort, propensity-score-matching was used to control selection bias. Results In total, 247 patients were included (A-T = 40.5%, A-TCb = 59.5%). After propensity-score-matching, pCR was significantly higher for A-TCb vs A-T (51.9% vs 34.2%, multivariate: OR = 2.40, P = 0.01). No differences in grade ≥3 haematological toxicities were observed. TILs increased from baseline to RD in the overall population and across A-T/A-TCb subgroups. TIL increase from baseline to RD was positively and independently associated with distant disease-free survival (multivariate: HR = 0.43, P = 0.05). Conclusions We confirmed in a clinical practice setting of TNBC patients receiving A-T NACT that the incorporation of weekly Cb significantly improved pCR. In addition, A-T +/− Cb enhanced immune infiltration from baseline to RD. Finally, we reported a positive independent prognostic role of TIL increase after NACT exposure.

Although 1% is the recommended cut-off to define estrogen receptor (ER) positivity, a 10% cut-off is often used in clinical practice for therapeutic purposes. We here evaluate clinical outcomes according to ER levels in a monoinstitutional cohort of non-metastatic triple-negative breast cancer (BC) patients undergoing (neo)adjuvant chemotherapy. Clinicopathological data of 406 patients with ER < 10% HER2-negative BC treated with (neo)adjuvant chemotherapy between 01/2000 and 04/2019 were collected. Patients were categorized in ER-negative (ER < 1%; N = 364) and ER-low positive (1–9%, N = 42). At a median follow-up of 54 months, 88 patients had relapsed and 64 died. No significant difference was observed in invasive relapse-free survival (iRFS) and overall survival (OS) according to ER expression levels, both at univariate and multivariate analysis (5-years iRFS 74.0% versus 73.1% for ER-negative and ER-low positive BC, respectively, p = 0.6; 5-years OS 82.3% versus 76.7% for ER-negative and ER-low positive BC, respectively, p = 0.8). Among the 165 patients that received neoadjuvant chemotherapy, pathological complete response rate was similar in the two cohorts (38% in ER-negative, 44% in ER-low positive, p = 0.498). In conclusion, primary BC with ER1–9% shows similar clinical behavior to ER 1% BC. Our results suggest the use of a 10% cut-off, rather than <1%, to define triple-negative BC.

Abemaciclib is approved for HR+/HER2- metastatic breast cancer in combination with endocrine therapy (ET). We evaluated safety and efficacy of abemaciclib in association with ET in a single-institution cohort. Patients with HR+/HER2- metastatic breast cancer treated with abemaciclib in combination with ET as first or second line between April 2019 and November 2020 at Istituto Oncologico Veneto of Padova were identified. Clinicopathological characteristics, adverse events and their grade (according to CTCAE 5.0 criteria) were collected. Objective response rate (ORR) according to RECIST 1.1 was also evaluated. 72 patients were included: 52 received abemaciclib as first line, 20 as second line; in 49 patients abemaciclib was administer in combination with an aromatase inhibitor, in 23 with fulvestrant. 95.8% of patients experienced at least one adverse event. The most common was diarrhea (79.2%), mainly G1 (66.7% of cases). Other common toxicities were: neutropenia (56.9%), increased serum creatinine (38.9%), anemia (37.5%), nausea (34.7%), fatigue (23.6%) and hypertransaminasemia (22.2%). Toxicities were mainly low grade; 10.1% of adverse events where G3-4. 48.6% of patients required a temporary interruption of abemaciclib and 45.8% a dose reduction. All 17 patients aged ≥70 required a dose reduction. A concomitant palliative radiotherapy was administered in 16 patients: in all cases radiotherapy was regularly completed; a temporary interruption of abemaciclib was required in 3 patients due to hematologic toxicities. In patients with measurable disease, ORR was 55.7%, significantly higher in first line compared to second line (70.5% vs 17.7%, p<0.001). ORR was not significantly different between full and reduced dose (46.9% vs 65.5%, p=0.143). At the time of the present analysis, 51 patients are still on therapy. Treatment discontinuations were due to disease progression in 17 patients, and to adverse events in 4 (G3 increased ALT in 1 patient and persistent G2-3 cutaneous toxicity in 3 patients). In a real-world population, abemaciclib was associated with a meaningful rate of objective response, with a favourable safety profile. Side effects were mostly low grade, manageable with temporary interruptions or dose reductions.

Abstract Background: Tumor infiltrating lymphocytes (TILs) are strong prognostic biomarkers for early TNBC. We evaluated the role of CD8, FOXP3 and PD-L1 expression in refining prognostic models for non-metastatic TNBC in a large cohort of patients treated with standard therapy. Methods: Consecutive patients diagnosed with stage I-III TNBC (ER/PgR <10%, HER2 0/1+ or ISH non amplified) between May 2012 and December 2015 were included. All patients received treatment with surgery, chemotherapy (neoadjuvant or adjuvant) and radiotherapy (when indicated). For each case, three FFPE tumor slides were stained for CD8 (Clone C8/144B, Dako Cytomation), FOXP3 (Clone 236A/E7, dilution 1:200, Abcam) and PD-L1 (IHC 73-10 Research Use Only assay developed by Agilent Technologies). One slide was stained for cytokeratins with MNF116. Digital slides were evaluated by a Visiopharm® software application, following alignment of the CD8, FOXP3 and PD-L1 slides with the MNF116 slide. The density of CD8 and FOXP3 expression was calculated as the number of cells/mm2 of stroma area. For PD-L1, the % of positive stromal cells over the total of stromal cells was analyzed. Disease-free survival (DFS) was calculated from diagnosis to relapse or death. The Harrell’s c-index was used to determine the cut-offs for CD8, FOXP3 and PD-L1 to be used in survival analyses. Results: 265 TNBC pts were evaluated. Median TILs was 10% (Q1-Q3 3%-25%), median CD8 was 249 (Q1-Q3 109-568), median FOXP3 was 57 (Q1-Q3 21-134), median PD-L1 was 5.2% (Q1-Q3 0.2%-25.4%). TILs, CD8, FOXP3 and PD-L1 were positively correlated with each other (p<0.001): CD8 showed strong correlation with TILs (Spearman’s coefficient 0.753), FOXP3 and PD-L1 showed moderate correlation with TILs (Spearman’s coefficient 0.535 and 0.587). Higher TILs, CD8, FOXP3 and PD-L1 were associated with age ≤50yrs (p=0.002, p=0.004, p=0.065, p=0.011), Grade 3 (p=0.001, p=0.006, p=0.006, p=0.003) and Ki67 ≥30% (p=0.056, p=0.024, p=0.004, p=0.005). There was no association between immune markers and stage. Among classic clinicopathologic factors, TILs (10% increments) and stage at diagnosis were independent prognostic parameters in multivariate analysis (HR 0.81, 95% CI 0.69-0.94 p=0.005 for TILs, HR 2.01 95% ci 1.01-4.23 P=0.047 for stage II vs stage I and HR 5.31 95% CI 2.54-11.11 p<0.001 for stage III vs stage I). In univariate analysis, high CD8 (≥443), high FOXP3 (≥57) and high PD-L1 (>20%) were all significantly associated with improved DFS (HR 0.36 95%CI 0.18-0.72, p=0.004 for CD8; HR 0.48 95%CI 0.28-0.80, p=0.005 for FOXP3; HR 0.52 95%CI 0.28-0.97, p=0.039 for PD-L1). FOXP3 and PD-L1 provided significant additional prognostic information beyond a model containing TILs and stage: likelihood ratio χ2 5.12, p=0.024 for FOXP3; likelihood ratio χ2 5.52, p=0.019 for PD-L1. CD8 did not add relevant prognostic information beyond TILs and stage (likelihood ratio χ2 2.76, p=0.097). Including both FOXP3 and PD-L1 did not add further prognostic information to models already containing TILs, stage and either FOXP3 or PD-L1. Conclusions: FOXP3 and PD-L1 expression evaluated with a software-assisted method were prognostic for stage I-III TNBC pts treated with standard therapy and may contribute to refine the prognostic stratification beyond stage and TILs. This study was supported by a grant from Merck KGaA. Citation Format: Maria Vittoria Dieci, Vassilena Tsvetkova, Gaia Griguolo, Federica Miglietta, Deborah Bacchin, Giulia Tasca, Carlo Alberto Giorgi, Enrico Cumerlato, Enrico Orvieto, Valentina Guarneri, Pierfranco Conte. Integrating CD8, FOXP3 and PD-L1 expression in prognostic models for triple negative breast cancer (TNBC): An analysis of 265 patients treated with standard therapy for stage I-III disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-14.

BACKGROUND:Tumour infiltrating lymphocytes (TILs) are an established prognostic biomarker for triple-negative breast cancer (TNBC). We evaluated the role of programmed cell-death ligand-1 (PD-L1), CD8 and FOXP3 expression in refining a prognostic model for non-metastatic TNBC beyond classic factors and TILs. METHODS:Primary tumour samples from 244 early patients with TNBC, all treated with surgery and chemotherapy, were collected. Stromal TILs were evaluated on haematoxylin-eosin slides according to guidelines. PD-L1, CD8 and FOXP3 were assessed by immunohistochemistry and evaluated by digital pathology. RESULTS:TILs, PD-L1, CD8 and FOXP3 were positively correlated with each other (P < 0.001). TILs were confirmed as an independent prognostic factor. When PD-L1, CD8 and FOXP3 were added to multivariable models including classic factors (age, stage, histologic grade) and TILs, PD-L1 provided the largest amount of additional prognostic information: likelihood ratio χ2 4.60, P = 0.032 (in a model including classic factors and TILs 10% increments) and likelihood ratio χ2 6.50, P = 0.011 (in a model including classic factors and TILs >30% versus <30%). In the subset of patients treated with neoadjuvant chemotherapy, FOXP3 provided further prognostic information beyond classic factors, TILs and pathological complete response (pCR) (likelihood ratio χ2 5.01, P = 0.025). For patients who did not achieve a pCR, the expression of CD8 and PD-L1 was significantly increased from baseline to residual disease. CONCLUSIONS:Beyond clinicopathological factors and TILs, other immune biomarkers may add prognostic information for early TNBC. The increased PD-L1 expression on residual disease after neoadjuvant chemotherapy strengthens the rationale of testing immune checkpoint inhibitors in the post-neoadjuvant setting.

The human gut microbiome has been shown to influence the efficacy of anticancer therapies. Little is known regarding the role of gut microbiome in triple negative breast cancer patients receiving neoadjuvant chemotherapy. In this pilot prospective study, we characterized the gut microbiome of 18 triple negative breast cancer patients undergoing neoadjuvant chemotherapy. All patients received anthracycline and taxane-based chemotherapy, including carboplatin in 16 patients. Pre-treatment fecal samples were analyzed by 16S RNA sequencing. Shannon index was used to evaluate alpha-diversity. Differentially abundant bacterial OTUs between groups were determined using Linear Discriminant Analysis (LDA) Effect Size (LEfSe). Patients characteristics were as follows: premenopausal 44%, ductal infiltrating carcinoma 100%, histologic G3 100%, cN+ 67%, cT>2cm 89%. No significant association between Shannon diversity index and baseline characteristics was shown. As expected, the most abundant taxa at the phylum level were Bacteroidetes and Firmicutes. Ten patients achieved a pCR. The Shannon diversity index was significantly higher in pCR vs non-pCR patients: median 5.057 (95% CI 4.923-5.339) vs 4.639 (95% CI 4.427-4.913), p=0.016. At the genus level, Alistipes and Ruminococcaceae UCG-002 were significantly enriched in pCR vs non-pCR patients (LDA score >3.0, p<0.05). The results of this pilot study show preliminary insights into the potential implications of gut microbiome in neoadjuvant chemotherapy efficacy for triple negative breast cancer. Patients recruitment is ongoing, updated results will be presented including association between intestinal microbiome and tumor immune microenvironment.

Abstract Background: Targeting the PD-L1/PD-1 axis has proved to be effective in various cancers, including promising data for metastatic TNBC pts. The evaluation of PD-L1 expression is limited by the lack of standardized methods. Here we sought to evaluate the prognostic role of PD-L1 expression in a large cohort of patients with non-metastatic TNBC treated with standard therapy. Methods: Consecutive patients diagnosed with stage I-III TNBC (ER and PgR <10%, HER2 0/1+ or ISH non amplified) between May 2012 and December 2015 and treated at the Istituto Oncologico Veneto of Padova were included. All patients received treatment with surgery, chemotherapy (neoadjuvant or adjuvant) and radiotherapy (when indicated). For each case, one FFPE tumor slide was stained for PD-L1 with the PD-L1 IHC 73-10 Research Use Only assay developed by Agilent Technologies and one slide was stained for cytokeratins with MNF116 (to distinguish MNF116+ tumor cells from MNF116- stromal cells). Digital slides were evaluated by a specifically-developed Visiopharm® software application. Following alignment of the PD-L1 and MNF116 digital slides, the software analyzed PD-L1 expression on tumor cells (% of positively stained tumor cells/total tumor cells) and stromal cells (% of positively stained stromal cells/total stromal cells). Disease-free survival (DFS) was calculated from diagnosis to relapse or death. In survival analyses, PD-L1 was evaluated as continuous and categorical variable. Results: 265 TNBC pts were evaluated. Median PD-L1 was 2.6% (Q1-Q3 0%-18.6%) on tumor cells and 5.2% (Q1-Q3 0.2%-25.4%) on stromal cells. PD-L1 levels on tumor and stromal cells were positively correlated (spearman's 0.938, p<0.001). For further analyses, PD-L1 on stromal cells was considered. Higher PD-L1 was associated with age <50 yrs (p=0.011), Grade 3 (p=0.003) and Ki67 >30% (p=0.005). Lower PD-L1 was observed in lobular and apocrine tumors (p=0.001).Cox model for DFS showed HR=0.99 (95%CI 0.97-1.00, p=0.059) for every 1% PD-L1 increment. 3-yrs DFS was 86% for pts with PD-L1>20% (n=88, 29%) vs 75% for pts with PD-L1<20% (n=177, 71%): HR 0.52, 95%CI 0.28-0.97, p=0.039. PD-L1 at 20% cut-off maintained prognostic value in multivariate model including stage (HR 0.48, 95%CI 0.25-0.89, p=0.021). Of the 265 pts included, 108 received neoadjuvant chemotherapy (NACT). Of the 78 pts with residual disease after NACT, 61 had pre- and post-NACT samples evaluable for PD-L1. PD-L1 increased from pre- to post-NACT: median 2.7% (Q1-Q3 0%-26.9%) vs 20.1% (Q1-Q3 5.9%-41.4%), p<0.001. Pts with PD-L1>20% post-NACT showed improved DFS: 3-yrs DFS 68% vs 43% (HR 0.44, 95%CI 0.20-0.96, p=0.039), whereas PD-L1 pre-NACT did not show significant association with DFS in this subgroup (HR 0.47, 95%CI 0.23-1.40, p=0.218). Conclusions: PD-L1 expression evaluated with a software-assisted method was prognostic for stage I-III TNBC pts treated with standard therapy. The significant increase of PD-L1 on residual disease post-NACT supports the rationale to evaluate the efficacy of anti-PD-L1 drugs in this high-risk population. Citation Format: Dieci MV, Orvieto E, Tsvetkova V, Griguolo G, Miglietta F, Bonaguro S, Tasca G, Giorgi CA, Cumerlato E, Guarneri V, Conte P. PD-L1 expression and prognosis in triple negative breast cancer (TNBC): An analysis of 265 patients (pts) treated with standard therapy for stage I-III disease [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 P4-08-04.

Background: Neoplastic pericardial effusion (NPE) is a life-threatening condition that can worsen clinical outcome in cancer patients. The optimal management of NPE has yet to be defined because randomized studies are lacking. Objective: We report a retrospective monoinstitutional experience describing characteristics, management and prognostic factors in NPE patients. Design: We reviewed clinical, pathological, and echocardiographic features, therapeutic strategies, and outcome in NPE patients referred to our institute from August 2011 to December 2017. Measurements: Twenty-nine patients with NPE from solid tumors have been identified: 21 lung, 5 breast, and 3 other cancer patients. Results: Median age was 62 years. Most of the patients had Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≥2 (69%) and a symptomatic NPE (69%). In 52% of patients NPE was detected at first diagnosis of metastatic disease, and in 20% of patients pericardium was the only site of metastases. Most of the patients (62%) received systemic therapy, 28% received combined locoregional and systemic therapy, and 10% received locoregional therapy alone. Median overall survival (OS) from NPE diagnosis was 3.9 months. Patients with PS ≥2 had worse OS than patients with better PS <2 (hazard ratio [HR] 3.56, IC 95% 1.19-10.65, p 0.02). Older age, extrapericardial disease, and NPE at progression showed a trend of association with worse OS. Patients treated with locoregional therapy alone showed the shortest median OS (p 0.05). Conclusions: NPE is related to dismal prognosis. Poor PS significantly worsens survival and influences therapeutic approaches. Randomized studies are required to investigate prognostic factors and appropriate clinical management for patients with NPE.

Background: We evaluated immunohistochemical AR expression and correlation with prognosis in a large series of homogeneously treated patients with primary TNBC. Material and Methods: Patients diagnosed with stage I-III TNBC between 2000 and 2015 at Istituto Oncologico Veneto who received treatment with surgery and neoadjuvant and/or adjuvant chemotherapy were included. Whole tissue slides were stained for AR. AR-positive expression was defined as >1% of positively stained tumor cells. Distant-disease-free survival (DDFS) was calculated from diagnosis to distant relapse or death. Late-DDFS was calculated from the landmark of 3 years after diagnosis until distant relapse or death. Results: We included 263 primary TNBC patients. Mean AR expression was 14% (range 0-100%), and 29.7% (n = 78) of patients were AR+. AR+ vs. AR- cases presented more frequently older age (p < 0.001), non-ductal histology (p < 0.001), G1-G2 (p = 0.003), lower Ki67 (p < 0.001) and lower TILs (p = 0.008). At a median follow up of 81 months, 23.6% of patients experienced a DDFS event: 33.3% of AR+ and 19.5% of AR- patients (p = 0.015). 5 years DDFS rates were 67.2% and 80.6% for AR+ and AR- patients (HR = 1.82 95%CI 1.10-3.02, p = 0.020). AR maintained an independent prognostic role beyond stage, but when TILs were added to the model only stage and TILs were independent prognostic factors. AR was the only factor significantly associated with late-DDFS: 16.4% of AR+ and 3.4% of AR- patients experienced a DDFS after the landmark of 3 years after diagnosis (p = 0.001). Late-DDFS rates at 5 years from the 3-year landmark were 75.8% for AR+ and 95.2% for AR- patients (log-rankp P < 0.001: HR = 5.67, 95%CI 1.90-16.94, p = 0.002). Conclusions: AR expression is associated with worse outcome for patients with TNBC. In particular, AR+ TNBC patients are at increased risk of late DDFS events. These results reinforce the rationale of AR targeting in AR+ TNBC.