Background - In the NeoTRIPaPDL1 phase III trial, triple-negative breast cancer (TNBC) patients were randomized to receive nab-paclitaxel/carboplatin for 8 cycles (CT arm) with or without atezolizumab (CT/A arm). We previously reported that the majority of patients (65%) with PD-L1- at baseline converted to PD-L1+ (the majority with IC2/IC3) after the first cycle of treatment in the atezolizumab arm. Here we studied treatment associated changes in the molecular tumour features and immune microenvironment by PD-L1 expression. Methods – A total of 158 (56.4%) of patients enrolled in the NeoTRIPaPDL1 trial (76 and 82 from CT/A and CT arm, respectively) were included in this analysis by satisfying the following criteria: i) availability of a paired core biopsy at baseline and at day 1 of the second treatment cycle (D1C2); ii) evaluation of stromal TILs (sTILs) and staining for PD-L1 (Ventana SP142); iii) successful RNA-seq gene expression profiling. PD-L1 groups were defined as IC0/IC1 (<5% PD-L1low) vs IC2/IC3 (>=5% PD-L1high). Presence of different immune cell populations was quantified by gene expression profile deconvolution using ConsensusTME R package. Cancer hallmark gene set collection and custom signature activation status were estimated in each sample using singscore R package. Differences in score distribution were evaluated by 2-sided t-test. Results At baseline, both sTILs and immune cell signatures were upregulated in PD-L1high compared to PD-L1low tumours (p<0.001). No significant differences were found between the two treatment arms in the PD-L1high subpopulation. In the PD-L1low cases, both sTILs and immune related signatures were slightly downregulated in the CT/A arm (p<0.05), suggesting a modest unbalance among treatment arms with CT arm being slightly more inflamed than CT/A arm.At D1C2, PD-L1high tumours in the CT arm systematically had high sTILs (median=70%, range=30-90%), while PD-L1low tumours receiving CT/A had low sTILs in a significant proportion of cases (median=30%, range=0-90%; p<0.001). Similarly, at D1C2 several gene expression-estimated immune cell populations and immune-related signatures were upregulated in the CT arm compared to CT/A arm in PD-L1high tumours, with the weakest association observed for M2 macrophages (p=0.058). No tumour-related signatures were differentially expressed among the two treatment arms within groups with PD-L1high, suggesting that different treatment. modulate PD-L1 by engaging a different immune mileau instead of modulating tumor related features.Considering PD-L1low groups at D1C2, in the CT arm 21 tumors had >30% sTILs (n=21/69, 30.4%), while in the CT/A arm only 2 had sTILs >30% (2/27, 7.4%) (p<0.001). Analysis of gene expression data identified IFN-related signatures as the most upregulated in the CT compared to CT/A arm in PD-L1low cases at D1C2 (p<0.05). Conclusions Integrated dynamic analysis of PD-L1 expression and gene expression data highlighted significant treatment-specific changes of the immune landscape according to PD-L1 expression, when this biomarker is assessed during treatment. This indicates that the immune milieu associated with PD-L1 status is strongly dependent on the time of assessment in relationship to treatment received. Such observation may explain why in the NeoTRIP trial, baseline PD-L1 but not on-treatment PD-L1 was predictive of pCR in CT/A arm. In addition, our findings could have implications related to the use of PD-L1 as a predictive biomarker in pre-treated patients, especially when assessed early on during treatment. Citation Format: Maurizio Callari, Chiun-Sheng Huang, Daniel Egle, Begoña Bermejo, Claudio Zamagni, Matteo Dugo, Marc Thill, Antonio Anton, Marco Barreca, Stefania Russo, Eva Maria Ciruelos, Richard Greil, Stefania Zambelli, Balázs Gyorffy, Chanel Smart, Olivia Biasi, Pinuccia Valagussa, Giuseppe Viale, Luca Gianni, Giampaolo Bianchini. Immune milieu associated with PD-L1 status in TNBC is dependent on time of biomarker assessment and treatment received: A secondary analysis of the NeoTRIPaPDL1 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-02.

BACKGROUND:Chronic heart valve regurgitation induces left ventricular (LV) volume overload, leading to the development of hypertrophy and progressive dilatation of the ventricle to maintain physiological cardiac output. In order to prevent potential irreversible LV structural changes, the identification of the best timing for treatment is pivotal. OBJECTIVE:To assess the presence and extent of fibrosis in myocardial tissue in asymptomatic patients with valvular heart disease (VHD) and preserved LV dimensions and function undergoing cardiac surgery. METHODS:Thirty-nine patients were enrolled. Sixteen patients were affected by aortic or mitral regurgitation: they were all asymptomatic, undergoing valve surgery according to VHD European Society of Cardiology guidelines. Twenty-three patients with end-stage nonischemic dilated cardiomyopathy (DCM) and severe LV dysfunction undergoing cardiac surgery for implantation of a durable left ventricular assist device (LVAD) served as controls. During surgery, VHD patients underwent three myocardial biopsies at the level of the septum, the lateral wall and LV apex, while in LVAD patients the coring of the apex of the LV was used. For both groups, the tissue samples were analyzed on one section corresponding to the apical area. All slides were stained with hematoxylin and eosin and Masson's trichrome staining and further digitalized. The degree of fibrosis was then calculated as a percentage of the total area. RESULTS:Of 39 patients, 23 met the inclusion criteria: 12 had mitral or aortic insufficiency with a preserved ejection fraction and 11 had idiopathic dilated cardiomyopathy. Quantitative analysis of apical sections revealed a myocardial fibrosis amount of 10 ± 6% in VHD patients, while in LVAD patients the mean apical myocardial fibrosis rate was 38 ± 9%. In VHD patients, fibrosis was also present in the lateral wall (9 ± 4%) and in the septum (9 ± 6%). CONCLUSION:Our case series study highlights the presence of tissue remodeling with fibrosis in asymptomatic patients with VHD and preserved LV function. According to our results, myocardial fibrosis is present at an early stage of the disease, well before developing detectable LV dysfunction and symptoms. Since the relationship between the progressive magnitude of myocardial fibrosis and potential prognostic implications are not yet defined, further studies on this topic are warranted.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease, for which it is crucial to promptly detect actionable and prognostic alterations to drive specific therapeutic decisions, regardless of tumor resectability status. Endoscopic ultrasonography-guided fine-needle aspi-ration (EUS-FNA) is of key importance for PDAC diagnosis and can contribute significantly to tumor molecular profiling. METHODS: Comprehensive genomic profile by targeted next-generation sequencing (NGS) was performed on 2 independent PDAC patient cohorts. Cohort 1 consisted of 77 patients with resectable PDAC for whom the histologic sample at the time of resection was available; for 56 patients cytologic specimens at the time of diagnosis also were obtained by EUS-FNA. Cohort 2 consisted of 20 patients with unresectable PDAC, for whom only the EUS-FNA cytologic sample was available. RESULTS: In cohort 1, a complete concordant mutational profile between the cytologic sample at diag- nosis and the corresponding histologic specimen after surgery was observed in 88% of the cases, proving the ability to detect potential clinically relevant alterations in cytologic samples by NGS analysis. Notably, clinically actionable mutations were identified in 20% of patients. In cohort 2, comprehensive mutational profiling was obtained successfully for all samples. Consistent with the findings of cohort 1, KRAS, TP53, CDKN2A, and SMAD4 were the most altered genes. Most importantly, 15% of the patients harbored actionable mutations. CONCLUSIONS: Our findings show the feasibility of an NGS approach using both surgical specimens and cyto- logic samples. The model proposed in this study can be included successfully in the clinical setting for comprehensive molecular profiling of all PDAC patients irrespective of their surgical eligibility.

555 Background: In the NA-PHER2 study we assessed the association between biological pathways with pathological complete response (pCR) and Ki67 down-regulation Methods: Patients with centrally confirmed ER+ ( > 10%) HER2+ breast cancer (BC) were treated in two independent, non-randomized cohorts with neoadjuvant trastuzumab, pertuzumab, palbocilib with (Fulv, n = 30) or without (NoFulv, n = 28) fulvestrant (+/-LHRH analogues). We assessed RNA-seq on core-biopsies obtained pre-treatment [n = 53/58 (91.4%)], at day 14 [n = 49/58 (84.5%)], and on residual disease at surgery [n = 42/45 (93.3%)]. We investigated biomarker dynamics and association with pCR or Ki67 down-regulation (centrally evaluated) at day 14 and at surgery. In the overall population and in each cohort, we primarily assessed three pre-defined biomarkers (ER-metagene [from OncotypeDX], a CD8-metagene and ERBB2 expression), and secondarily we explored a pre-defined list of genesets. Continuous and categorical (median cut-point) variables were evaluated. Results: In the biomarker population, pCR rate was 22.5% (28.6% and 16.0% in Fulv and NoFulv cohorts). At baseline, continuous CD8-metagene (OR 1.85 [1.12-3.06], p = 0.016) and ER-metagene (OR 0.56 [0.34-0.90], p = 0.016) associated with higher and lower pCR rate, respectively. High ERBB2 (above median) was marginally associated with pCR (OR 3.83 [0.90-16.3], p = 0.068). Only ER- and CD8-metagenes retained significance in multivariate analysis and were similarly predictive in both cohorts. Combining categorical variables, the groups with high-CD8/low-ER and low-CD8/high-ER had 61.5% and 0% pCR rate respectively, whereas low-CD8/low-ER and high-CD8/high-ER had similar 15% pCR (p = 0.001). The association was significant in both cohorts (p = 0.019 Fulv; p = 0.028 NoFulv). Dynamic assessment of the same biomarkers at day 14 did not improve prediction. Higher ER-metagene at baseline, but not CD8 and ERBB2, was associated with robust down-regulation of Ki67 at day 14 (Ki67 < 2.7%, complete cell cycle arrest) only in Fulv cohort (p = 0.016). ER-metagene also associated with retained Ki67 down-regulation (Ki67 < 10%) at surgery (p = 0.002). Alternative ER- and immune-related signatures provided very similar results. The comprehensive landscape of complex molecular dynamics and exploratory association with outcome will be presented. Conclusions: In ER+/HER2+ BC, low expression of ER-related and high expression of immune-related genes identified patients with very high likelihood of achieving pCR with a chemo-free regimen. In the fulvestrant cohort, the group with high ER-metagene, despite a lower pCR rate, had higher Ki67 down-regulation at day 14, which has been associated with long-term benefit in luminal tumors. These findings provide a potential tool for tailored de-escalation strategies.

Abstract Background. Although long considered a tumor suppressor gene, PML (promyelocytic leukemia) also plays tumor-promoting functions in specific contexts. In vitro and in vivo studies have demonstrated that PML is upregulated by HIF-1α transcriptional activation in triple-negative breast cancer (TNBC) cells and it is implicated in promoting metastasis downstream of HIF-1α. This pro-metastatic function of PML is inhibited by arsenic trioxide, a pharmacological compound currently in use in acute promyelocytic leukemia. However, the clinical relevance of PML expression in BC has not been extensively investigated. In this study, we evaluated the association of PML expression with clinic-pathological factors and outcome (pathological complete response -pCR- and risk of recurrence) in the ETNA trial, and risk of recurrence in the TCGA. Methods. In the ETNA study (NCT01822314) 695 patients with HER2-negative breast cancer (BC) were randomized to receive neoadjuvant paclitaxel or nab-paclitaxel followed by 4 cycles of an anthracycline regimen. In the ITT study population, the two treatments did not show significantly different rates of pCR nor different Event-Free Survival (EFS) (Gianni JAMA Oncol 2018, Gianni ASCO 2019). A central histologic assessment of ER, PgR, HER2 status and Ki67 was mandatory. We evaluated PML expression by immunohistochemistry using the continuous histoscore (H-score) on pre-treatment core biopsies. The H-score is generated from the estimation of the percentage of cells with no (0), light (1+), moderate (2+) and strong (3+) intensity staining, and the corresponding score is generated with the following algorithm: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. We evaluated the association of PML with clinic-pathological features and clinical outcomes (pCR and EFS) in triple negative (TN) and in LuminalB-like (ER+ and/or PgR+, Ki67≥14%) groups. We also investigated the association between PML mRNA expression (RNA-seq) and DFS in HER2-negative BC (TN, n=192; ER+/HER2-, n=702) in the TCGA dataset. Results. In the ETNA study, PML was successfully assessed and valuable in 491 pts (70.6%). The average PML expression was 126.3 (median 120, range 0-295). In the ETNA trial, TNBC showed the highest expression of PML (p<1.0E-10). Within LumB-like tumors, the group with intermediate proliferation (Ki67 14-20%, LumB-int) had higher PML expression than the high proliferation group (Ki67>20%, LumB-high) (p=0.0005). However, within the LumB-high group, higher proliferation (Ki67>40%) showed a higher expression of PML (p=0.025), suggesting a non-linear relationship between PML and proliferation in luminal tumors. In LumB-high, PML was higher in PgR-negative tumors (p=1.0E-5). Finally, PML showed a positive association with higher stromal tumor-infiltrating lymphocytes (sTILs) both in LumB-like and TN group (p=0.019 and p=0.001, respectively). PML expression was not significantly associated with pCR and risk of recurrence in LumB-like nor in TN BC.In the TCGA dataset also PML expression was highest in the TN group (p<1.0E-10). No association was found between PML expression and risk of recurrence in any molecular subgroup. Conclusions. PML expression assessed by IHC in the ETNA trial was not predictive of likelihood of pCR nor of risk of recurrence in LumB-like and TN breast cancer. PML mRNA expression was not prognostic in TN and LumB-like tumors also in the TCGA dataset. Some interesting associations with biological features emerged which warrants biological interpretation of the complex role of PML in breast cancer. Supported in part by an unrestricted grant from Celgene Sarl, Swizerland Citation Format: Stefania Zambelli, Chanel Smart, Giampaolo Bianchini, Isabella Sassi, Mauro Mansutti, Antonio Anton, Lourdes Calvo, Giancarlo Bisagni, Begona Bermejo, Martina Uggè, Barbara Galbardi, Vladimir Semiglazov, Marc Thill, Jose Ignacio Chacon, Arlene Chan, Serafin Morales Murillo, Isabel Alvarez, Ainhara Lahuerta, Patrizia Zucchinelli, Claudio Doglioni, Pinuccia Valagussa, Ignasi Tusquets, Luca Gianni, Rosa Bernardi. Prognostic and predictive value of PML in the ETNA study and the TCGA [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 P5-06-21.

Background Genetic variants identified through genome-wide association studies (GWAS) are predominantly non-coding and typically attributed to altered regulatory elements such as enhancers and promoters. However, the contribution of non-coding RNAs to complex traits is not clear. Results Using targeted RNA sequencing, we systematically annotated multi-exonic non-coding RNA (mencRNA) genes transcribed from 1.5-Mb intervals surrounding 139 breast cancer GWAS signals and assessed their contribution to breast cancer risk. We identify more than 4000 mencRNA genes and show their expression distinguishes normal breast tissue from tumors and different breast cancer subtypes. Importantly, breast cancer risk variants, identified through genetic fine-mapping, are significantly enriched in mencRNA exons, but not the promoters or introns. eQTL analyses identify mencRNAs whose expression is associated with risk variants. Furthermore, chromatin interaction data identify hundreds of mencRNA promoters that loop to regions that contain breast cancer risk variants. Conclusions We have compiled the largest catalog of breast cancer-associated mencRNAs to date and provide evidence that modulation of mencRNAs by GWAS variants may provide an alternative mechanism underlying complex traits.

555 Background: We assessed TILs and PDL1 expression before, during and after neoadjuvant treatment in TNBC patients enrolled in the ETNA study, and investigated associations with clinical outcome. Methods: In ETNA patients randomly received paclitaxel or nab-paclitaxel followed by 4 cycles of an anthracycline regimen, including 219 centrally confirmed TNBC. We successfully measured stromal and intratumoral TILs (sTILs, iTILs) and PDL1 status (Ventana SP142, IC≥1%) on biopsies before [n = 186/213 (84.9%)], on d1 cycle 2 (d1c2) of therapy [n = 41/186 (22.0%)], and at surgery [SX, n = 65/129 (34.9%)]. We investigated the expression and modulation over time of TILs and PDL1 and their association with pCR and event-free survival (EFS). Results: Prevalence of PDL1+ was 35.5% (baseline), 20.6% (d1c2) and 30.1% (SX). At each time-point sTILs and iTILs were higher in PDL1+ cases (p≤0.01). An effect of age of the tumor blocks (5-7.5 years) or pre-analytical issues could not be ruled out for the relatively low rate of PDL1 positivity. Paired PDL1 at baseline and d1c2 showed conversion in 25.7% (pos to neg [11.4%] or neg to pos [14.3%]). Comparing PDL1 at baseline and SX, the conversion rate was 30% (pos to neg [8.3%] or neg to pos [21.6%]). sTILs and iTILs significantly increased at cycle 2, more significantly in pCR (p≤0.001) than in RD (p≤0.05) cases, and a not significant trend of decrease was observed at surgery PDL1+ tumors had a higher pCR rate (54.7% vs 32.5%, p = 0.004). PDL1 retained significance (OR 2.00 [1.04-3.88], p = 0.039) after adjustment for sTILs (OR 1.21 [1.03-1.42], p = 0.021). High iTILs and sTILs at d1c2, but not PDL1 status, were predictive of pCR. Notably, adjusting for sTILs, PDL1+ tumors at d1c2 showed a trend for association with lower pCR rate (OR 0.06 [0.01-1.15], p = 0.062). sTILs at cycle 2 was the most informative variable (OR 1.61 [1.28-1.61], p = 0.004) and provided independent information to baseline biomarkers. Baseline PDL1 and biomarkers at cycle 2 were not associated with EFS. In surgical samples with RD, higher sTILs, but not iTILs and PDL1 status, were associated with a trend for a lower risk of recurrence (HR 0.19 [0.02-1.39], p = 0.068). Conclusions: sTILs assessment on core biopsies after one cycle of taxane is a promising early biomarker of pCR. PDL1, as well as sTILs and iTILs, provided independent prediction of pCR and were strongly modulated by treatment. The modulation of PDL1 expression should be considered whenever PDL1 is assessed in view of identifying candidates to atezolizumab in 1 st line advanced setting.

Abstract Background. We investigated the association between TILs assessed by different metrics and clinical outcome (pathological complete response -pCR- and risk of recurrence) in the ETNA trial. Methods. In the ETNA study (NCT01822314) 695 patients with HER2-negative breast cancer (BC) were randomized to receive neoadjuvant paclitaxel or nab-paclitaxel followed by 4 cycles of an anthracycline regimen. In the ITT study population, the two treatments did not show significantly different rates of pCR nor different Event-Free Survival (EFS) (Gianni JAMA Oncol 2018, Gianni ASCO 2019). We measured TILs by optical evaluation on pre-treatment core biopsies using three different metrics: stromal and intratumoral TILs (sTILs, iTILs), and hotspot TILs (hTILs)ie. the field of highest TILs density, in order to capture some of the spatial heterogeneity of TILs reported as more informative in ER+ BCs (Heindl JNCI 2018). We then investigated the association of TILs with pCR and EFS separately in TN and in LuminalB-like (ER+ and/or PgR+, Ki67≥14%) groups, as well as possible treatment-biomarker interactions. Results. TILs could be assessed in 589 pts (85%). The three TILs metrics were significantly correlated (p<10E-10) (sTILs vs iTILs, cor 0.59; sTILs vs hTILs, cor 0.83; iTILs vs hTILs, cor: 0.57). The correlation between hTILs and sTILs was lower for LumB-like tumors and lower TILs levels. iTILs were less informative than the other two metrics.Higher sTILs and hTILs as continuous variable (10% increase) were significantly associated with likelihood of pCR in both tumor subtypes (all p<0.001). In TN tumors sTILs and hTILs provided similar and not independent predictive value (OR 1.23 [1.10-1.37], p=0.002 and OR 1.34 [1.14-1.58], p=0.0005). In LumB-like tumors hTILs were slightly more predictive of pCR than sTILs. A 10% increase in hTILs was associated with a significant increase of pCR rate (OR 1.43 [1.28-1.61], p=1E-09). In addition, hTILs were more informative than sTILs (Likelihood Ratio Test p=0.01 for the addition of hTILs to sTILs in the model). In the TN BC group, higher expression sTILs and hTILs was associated with lower risk of recurrence, with HR 0.90 [0.81-0.99], p=0.041 and HR 0.84 [0.71-0.99], p=0.044, respectively, but in LumB-like BC they were not associated with EFS.We also evaluated the interaction between TILs and treatment benefit as exploratory analysis. The test for interaction was negative (p=0.251) in TN BC, while in the LumB-like group the test for interaction for hTILs and sTILs as continuous variable was 0.024 and 0.10, respectively. The test for interaction for sTILs was also significant when the transformed log variable was used (p=0.043), indicating a possible non-linear effect. To better evaluate the quality of this interaction, we applied an arbitrary cut-off to hTILs to define two groups: low (≤ 20%, n=282) and high (>20%, n=126) TILs. In the low hTILs group, abraxane was significantly more effective than paclitaxel (HR 0.64 [0.26-0.89], p=0.02). In the high hTILs group, paclitaxel was associated with better long-term outcome (HR 3.79 [1.33-10.8], p=0.01). If the same cut-off was applied in TN BC, abraxane had a numerical trend for superiority compared to paclitaxel in the low hTILs tumors (HR 0.64 [0.34-1.20]). No differences were observed in the high TIL group. Conclusions. Pre-treatment assessment of TILs in the ETNA trial was predictive of the likelihood of pCR regardless of the tumor subtype, and was prognostic in the TN subgroup. The assessment of hotspot TILs seems to be more informative than average stromal TILs in LumB-like tumors. Finally, different levels of hTILs at baseline were associated with different benefit from abraxane compared to paclitaxel. This finding warrants independent confirmation, and the mechanism for the association is being investigated. Supported in part by an unrestricted grant from Celgene Sarl, Swizerland Citation Format: Giampaolo Bianchini, Chanel Smart, Mauro Mansutti, Antonio Anton, Luca Licata, Isabella Sassi, Lourdes Calvo, Giancarlo Bisagni, Begona Bermejo, Vladimir Semiglazov, Marc Thill, Jose Ignacio Chacon, Arlene Chan, Serafin Morales Murillo, Isabel Alvarez, Ainhara Lahuerta, Patrizia Zucchinelli, Claudio Doglioni, Giuseppe Viale, Pinuccia Valagussa, Ignasi Tusquet, Luca Gianni. Clinical implication of tumor infiltrating lymphocytes (TILs) in the ETNA study [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 PD5-05.

Abstract Background We assessed TILs before and during treatment in patients with ER+/HER2+ breast cancer (BC) enrolled in the NA-PHER2 study, and evaluated the association between TILs and their modulation over treatment with likelihood of achieving a pCR. Methods In the NA-PHER2 study (NCT02530424), patients with centrally confirmed ER+ (>10%) HER2+ BC were treated in two independent, non-randomized, cohorts with neoadjuvant trastuzumab, pertuzumab, palbocilib with (Cohort A, n=30) or without (Cohort B, n=28) fulvestrant (+/-LHRH analogues). We measured TILs by optical evaluation on core-biopsies obtained pre-treatment [pre-CBX, n=56/58 (96.5%)], at 14 days if invasive tumor cells [14d-CBX, n=52/53 (98.1%)], and on residual disease at surgery [SX, n=45/45 (100%)]. We scored TILs as stromal and intratumoral TILs (sTILs, iTILs), and hotspot TILs (hTILs) i.e. the fields of highest TILs density. We investigated the association between baseline and 14 days TILs with pCR, including the early TILs changes (pre vs 14 days) [delta TILs (ΔTILs) was the absolute percentage change). TILs biomarkers were assessed as continuous variable (10% increase). Results The three TILs metrics were significantly correlated at all time-points (all p<1E-04, all corr >0.55). TILs metrics were not associated with T and N stage, PgR status. sTILs were correlated with Ki67 as continuous variable at 14 days (corr 0.41 p=0.003) and surgery (corr 0.34, p=0.025), but not at baseline. At baseline (pre-CBX), only high sTILs were associated with pCR (OR 1.55 [1.01-2.37], p=0.044). At 14 days (on treatment), all TILs assessments were associated with pCR (OR 1.96 [1.16-3.31], p=0.011 for sTILs; OR 5.85 [1.39-24.6], p=0.016 for iTILs; OR 1.58 [1.16-2.14], p=0.004). When combined in multivariate model, they did not provide independent information. Considering early changes of TILs (baseline vs 14 days), only ΔiTILs was associated with pCR (OR 3.09 [1.50-6.38], p=0.002). When baseline, 14 days and dynamic biomarker was assessed in multivariate, only ΔiTILs retained the significance. The predictive value of TILs was similar in the two treatment cohorts. Overall, there was not average difference between any TILs assessment method comparing the three time points. By evaluating only patients with RD and TILs assessment available at all three time points (n=41/45, 91.1%), some intra-patient variability emerged. ΔTIL higher than 10% was observed in 13/41 (31.7%) cases. Seven (17.1%) had an increase, 4 (9.7%) a mixed behavior and 2 (4.9%) had a decrease. Dynamic change of TILs was not associated with change in Ki67. When TILs infiltration in the two treatment cohort was compared at different time points, it was different only at surgery, when sTILs and iTILs were lower in patients treated with Fulvestrant in Cohort A (p=0.19 and p=0.013, respectively). Conclusions In the NA-PHER2 study, we show that in ER+/HER2+ BC the likelihood of achieving pCR using a chemo-free regimen is larger in patients with higher sTILs infiltration. TILs assessment on core biopsies after 14 days was also predictive of pCR, and early changes of iTILs was the most informative predictor suggesting that treatment may facilitate T cell infiltration in agreement with the postulated contribution of CDK4/6 to immune evasion (Goel S Nature 2017, Jerby-Arnon L Cell 2018). No average modulation of immune biomarkers was found. However, individual pts modulation suggests that immune effect might be relevant also for some patients not achieving a pCR. Notably, the presence of endocrine treatment may contribute to dampen immune activation. Changes of Ki67 and TILs were not associated suggesting that they capture different biological phenomena. Supported in part by an unrestricted grant from Pfizer Italia S.r.l. and from Roche S.p.a. Italia Citation Format: Giampaolo Bianchini, Chanel Smart, Giancarlo Bisagni, Marco Colleoni, Mauro Mansutti, Claudio Zamagni, Lucia Del Mastro, Stefania Zambelli, Luca Licata, Antonio Frassoldati, Giuseppe Viale, Pinuccia Valagussa, Luca Gianni. Modulation of tumor infiltrating lymphocytes (TILs) by palbociclib, trastuzumab, pertuzumab +/- fulvestrant in ER+/HER2+ patients (pts) enrolled in the Michelangelo NA-PHER2 trial [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 P4-14-01.

We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease.

AimsA better understanding of the expression of cancer/testis antigens (CTAs) in breast cancer might enable the identification of new immunotherapy options, especially for triple‐negative (TN) tumours, which lack expression of the conventional therapeutic targets oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The aim of this study was to quantify the expression of MAGE‐A and NY‐ESO‐1 CTAs in breast cancer, and relate this to known clinicopathological parameters.Methods and resultsWe surveyed MAGE‐A and NY‐ESO‐1 expression in an unselected cohort of 367 breast tumours (of which 65 were TN), with accompanying clinical follow‐up data, by using immunohistochemical analysis of tissue microarrays. Relevant to their potential as vaccine targets in breast cancer, MAGE‐A was expressed in 13% of cases, and NY‐ESO‐1 in 3.8%, with the majority of tumours showing fairly homogeneous staining within individual tissue cores (~85% of cases with staining in >75% of tumour cells). Most NY‐ESO‐1‐positive cases also expressed MAGE‐A (P = 2.06 × 10−9), and both were strongly associated with the TN phenotype (P < 0.0001), with the most proliferative and poorly differentiated cases, in paticular, showing genomic instability. This was characterised by coexpression of c‐Kit and TTK, and overexpression of p53.ConclusionsMAGE‐A and NY‐ESO‐1 are frequently expressed in TN breast cancer (~47% and 17% of TN cases, respectively), suggesting that targeting them could be feasible in this patient group. Expression is reasonably homogeneous in positive cases, suggesting that immunohistochemical analysis of tissue biopsies would be a reliable companion biomarker.

Cell lines are extremely useful tools in breast cancer research. Their key benefits include a high degree of control over experimental variables and reproducibility. However, the advantages must be balanced against the limitations of modelling such a complex disease in vitro. Informed selection of cell line(s) for a given experiment now requires essential knowledge about molecular and phenotypic context in the culture dish. We performed multidimensional profiling of 36 widely used breast cancer cell lines that were cultured under standardised conditions. Flow cytometry and digital immunohistochemistry were used to compare the expression of 14 classical breast cancer biomarkers related to intrinsic molecular profiles and differentiation states: EpCAM, CD24, CD49f, CD44, ER, AR, HER2, EGFR, E-cadherin, p53, vimentin, and cytokeratins 5, 8/18 and 19. This cell-by-cell analysis revealed striking heterogeneity within cultures of individual lines that would be otherwise obscured by analysing cell homogenates, particularly amongst the triple-negative lines. High levels of p53 protein, but not RNA, were associated with somatic mutations (p = 0.008). We also identified new subgroups using the nanoString PanCancer Pathways panel (730 transcripts representing 13 canonical cancer pathways). Unsupervised clustering identified five groups: luminal/HER2, immortalised ('normal'), claudin-low and two basal clusters, distinguished mostly by baseline expression of TGF-beta and PI3-kinase pathway genes. These features are compared with other published genotype and phenotype information in a user-friendly reference table to help guide selection of the most appropriate models for in vitro and in vivo studies, and as a framework for classifying new patient-derived cancer cell lines and xenografts.

BACKGROUND:The complex interaction between multiple cell types and the microenvironment underlies the diverse pathways to carcinogenesis and necessitates sophisticated approaches to in vitro hypotheses testing. The combination of mixed culture format with high content immunofluorescence screening technology provides a powerful platform for observation of cell type specific behavior.METHODS:We have developed a versatile, high-throughput method for assessing cell-type specific responses. In addition to the specificity and sensitivity offered traditionally by immunofluorescent detection in flow cytometry, the 'in-cell' analysis method we describe provides the added benefits of higher throughput and the ability to analyse protein subcellular localisation in situ. Furthermore, elimination of the cell dissociation step allows for more immediate analysis of responses to specific extrinsic stimuli. We applied this method to investigate ionising radiation treatment response in normal breast epithelial cells, measuring growth rate, cell cycle response and double-strand DNA breaks.RESULTS:The 'in-cell' analysis approach elucidated several interesting donor and cell-type specific differences. Notably, in response to ionizing radiation we observed differential expression in luminal and basal-like cells of a member of the APOBEC enzyme family, recently identified as a critical driver of an oncogenic signature. Our findings suggest that this enzyme is active in the normal breast epithelium during DNA damage response.CONCLUSIONS:We demonstrate the practical application of a new method for assessing cell-type specific change in mixed cultures, especially the analysis of normal primary cultures, overcoming a major technical issue of dissecting the response of multiple cell types in a heterogeneous population.

Regulation of Ca 2+ transport is vital in physiological processes, including lactation, proliferation and apoptosis. The plasmalemmal Ca 2+ pump isoform 2 (PMCA2) a calcium ion efflux pump, was the first protein identified to be crucial in the transport of Ca 2+ ions into milk during lactation in mice. In these studies we show that PMCA2 is also expressed in human epithelia undergoing lactational remodeling and also report strong PMCA2 staining on apical membranes of luminal epithelia in approximately 9% of human breast cancers we assessed. Membrane protein expression was not significantly associated with grade or hormone receptor status. However, PMCA2 mRNA levels were enriched in Basal breast cancers where it was positively correlated with survival. Silencing of PMCA2 reduced MDA-MB-231 breast cancer cell proliferation, whereas silencing of the related isoforms PMCA1 and PMCA4 had no effect. PMCA2 silencing also sensitized MDA-MB-231 cells to the cytotoxic agent doxorubicin. Targeting PMCA2 alone or in combination with cytotoxic therapy may be worthy of investigation as a therapeutic strategy in breast cancer. PMCA2 mRNA levels are also a potential tool in identifying poor responders to therapy in women with Basal breast cancer.

Calcium pumps and channels modulate cell proliferation and apoptosis by regulating intracellular calcium (Ca2+). The plasma membrane Ca2+ ATPase isoform, PMCA2, is a calcium efflux mechanism that extrudes Ca2+ from the cytosol into the extracellular space. PMCA2 has a restricted expression, including expression in cochlear hair cells and cerebellar Purkinje cells. PMCA2 expression is increased in mouse mammary glands during lactation where it plays a major role in the excretion of Ca2+ into milk; however, PMCA2 expression has not been assessed in human breast tissue exhibiting lactational changes. Our previous studies have shown that PMCA2 mRNA levels are elevated in some breast cancer cell lines and that pan-PMCA antisense attenuates the proliferation of MCF-7 breast cancer cells. However, the consequences of silencing PMCA2 in breast cancer cells are still not well understood. Our study assessed PMCA2 expression in breast tissue exhibiting lactational change and in human malignant breast tissue samples. The role of PMCA2 in the proliferation of breast cancer cells was also evaluated. Immunohistochemistry using a rabbit anti-PMCA2 antibody showed membranous PMCA2 expression in the luminal epithelium of breast tissue exhibiting lactational change. PMCA2 expression was assessed in human breast tumor samples assembled into tissue microarrays. Nine of 96 breast tumours (9.4%) showed membranous PMCA2 staining. PMCA2 expression did not significantly correlate with the breast cancer pathological markers, estrogen, progesterone or HER2 receptor status. High-content imaging demonstrated that PMCA2 silencing in MDA-MB-231 breast cancer cells is associated with a reduction in cell number and an inhibition of the percentage of S-phase positive cells. The effect of PMCA2 silencing combined with various cytotoxics (cisplatin, doxorubicin or mitomycin C) on cell proliferation was assessed in MDA-MB-231 cells using a kinetic imaging system (IncuCyte). The results showed that PMCA2 silencing promotes the effects of some cytotoxics. These findings indicate that PMCA2 protein expression is elevated during human lactation and in some breast cancers. Inhibitors of PMCA2 may represent a novel therapeutic strategy for some breast cancers.

Our understanding of breast cancer heterogeneity at the protein level is limited despite proteins being the ultimate effectors of cellular functions. We investigated the heterogeneity of breast cancer (41 primary tumors and 15 breast cancer cell lines) at the protein and phosphoprotein levels to identify activated oncogenic pathways and developing targeted therapeutic strategies. Heterogeneity was observed not only across histological subtypes, but also within subtypes. Tumors of the Triple negative breast cancer (TNBC) subtype distributed across four different clusters where one cluster (cluster ii) showed high deregulation of many proteins and phosphoproteins. The majority of TNBC cell lines, particularly mesenchymal lines, resembled the cluster ii TNBC tumors. Indeed, TNBC cell lines were more sensitive than non-TNBC cell lines when treated with targeted inhibitors selected based on upregulated pathways in cluster ii. In line with the enrichment of the upregulated pathways with onco-clients of Hsp90, we found synergy in combining Hsp90 inhibitors with several kinase inhibitors, particularly Erk5 inhibitors. The combination of Erk5 and Hsp90 inhibitors was effective in vitro and in vivo against TNBC leading to upregulation of pro-apoptotic effectors. Our studies contribute to proteomic profiling and improve our understanding of TNBC heterogeneity to provide therapeutic opportunities for this disease.

The transcription factor SOX2 is essential for maintaining pluripotency in a variety of stem cells. It has important functions during embryonic development, is involved in cancer stem cell maintenance, and is often deregulated in cancer. The mechanism of SOX2 regulation has yet to be clarified, but the SOX2 gene lies in an intron of a long multi-exon non-coding RNA called SOX2 overlapping transcript (SOX2OT). Here, we show that the expression of SOX2 and SOX2OT is concordant in breast cancer, differentially expressed in estrogen receptor positive and negative breast cancer samples and that both are up-regulated in suspension culture conditions that favor growth of stem cell phenotypes. Importantly, ectopic expression of SOX2OT led to an almost 20-fold increase in SOX2 expression, together with a reduced proliferation and increased breast cancer cell anchorage-independent growth. We propose that SOX2OT plays a key role in the induction and/or maintenance of SOX2 expression in breast cancer.