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Resume Introduction et but de l’etude La tolerance de l’association d’une radiotherapie concomitante a un traitement par pertuzumab et trastuzumab ) est encore inconnue. L’objectif de ce travail etait d’evaluer la tolerance de cette association chez des patientes traitees pour un cancer du sein localement evolue ou metastatique surexprimant HER2 . Materiel et methodes Etude retrospective menee a l’institut Curie chez toutes les patientes traitees en concomitance par pertuzumab, trastuzumab et irradiation. La radiotherapie a ete delivree pendant un traitement par maintenance par pertuzumab et trastuzumab a respectivement 420 mg (dose totale) et 6 mg/kg toutes les trois semaines sans chimiotherapie. L’evaluation de la toxicite a ete faite selon la « National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 ». La fraction d’ejection ventriculaire gauche (FEVG) a ete evalue dans le bilan pre-therapeutique et puis tous les 3 a 4 mois. Resultats et analyse statistique Nous avons etudie 77 patientes traitees entre octobre 2013 et decembre 2019 avec un suivi median de 38 mois (extremes : 0–246 mois). L’âge median etait de 53 ans (extremes : 36–86 ans). Il y avait 50 patientes (64,9 %) atteintes de metastatses au moment du diagnostic et 27 patientes (35,1 %) en situation de recidive. Toutes les patientes ont recu comme premiere ligne therapeutique une chimiotherapie a base de taxanes suivie par pertuzumab et trastuzumab avec un nombre de cycle median de 34 (10–85). Toutes les patientes avaient une reponse partielle ou complete selon les criteres RECIST. Les volumes de radiotherapie etaient le sein (41 patientes, 53,2 %) et la paroi thoracique (29 patientes, 37,7 %) a la dose de 50 Gy avec une duree mediane de traitement de 39 jours. Une irradiation des aires ganglionnaires regionales a ete delivree chez 53 patientes (68,8 %) comme suit : irradiation sus/sous claviculaire axillaire chez 52 patientes (67,5 %) et mammaire interne chez 31 patientes (40,3 %). Pour 20 patientes, la radiotherapie etait a visee palliative : osseuse (12 patientes, 15,6 %), de encephale in toto (2 patientes, 2,6 %), de nodules metastatiques cerebraux (six patientes). Comme toxicite aigue, nous avons observe : une radioepitheliite de grade 1 chez 36 patientes (46,8 %), de grade 2 chez 17 patientes (22,1 %) et de grade 3 chez trois patientes (3,9 %). Une patiente a souffert d'une œsophagite de grade 2 et une d'une baisse asymptomatique de la FEVG en cours de traitement par pertuzumab, trastuzumab et irradiation. Comme toxicite tardive, nous avons observe des telangiectasies chez deux patientes, de grade 1 (1,3 %) et de grade 2 (1,3 %) ; il y a eu un cas de cardiotoxicite de grade 3 huit mois apres la fin de la radiotherapie. Conclusion Le traitement concomitant par irradiation, pertuzumab et trastuzumab semble avec un bon profil de tolerance ; une etude prospective plus large avec un suivi plus prolonge reste cependant necessaire pour confirmer ces resultats.

Background Following the PALOMA-3 study results, the combination of palbociclib, a CDK4/6 inhibitor, with fulvestrant, a selective estrogen receptor degrader, has become a standard therapy in women with estrogen receptor-positive (ER+) HER2-negative (HER2−) metastatic breast cancer (MBC). Palbociclib has been shown to increase the progression-free survival (PFS) overall but no predictive biomarker of palbociclib efficacy has been validated so far. We thus evaluated whether early changes of circulating tumor DNA (ctDNA) levels are associated with palbociclib plus fulvestrant efficiency. Methods ER+ HER2− MBC patients were included in a prospective observational cohort before treatment initiation. Tumor response was assessed by radiological evaluation (RECIST v1.1) every 3 months. Plasma samples were collected before treatment (baseline), at day 15 (D15), at day 30 (D30), and at disease progression. We searched for somatic mutations from archived tumor tissues by targeted deep sequencing. For patients with somatic mutations identified, circulating tumor DNA (ctDNA) was tracked using digital droplet PCR. Ratios of ctDNA levels ([D15/baseline] and [D30/baseline]) were then correlated with prospectively registered patient characteristics and outcomes. Results Twenty-five of the 61 patients enrolled had a somatic mutation testable in plasma (NPIK3CA = 21, NTP53 = 2, NAKT1 = 2). At baseline, 84% of patients had detectable ctDNA levels but ctDNA levels had no prognostic impact on PFS (p = 0.10). Among those patients, ctDNA was still detected in 82% at D15 and 68% at D30. ctDNA clearance observed at day 30 was associated with longer PFS (HR = 7.2, 95% CI = 1.5–32.6, p = 0.004). On the contrary, a [D30/baseline] ctDNA ratio > 1 was associated with a shorter PFS (HR = 5.1, 95% CI = 1.4–18.3, p = 0.02) and all 5 patients with increased ctDNA levels at D30 showed disease progression after 3 months under palbociclib-fulvestrant. Finally, at the time of radiological tumor progression, ctDNA was detected in all patients tested. Conclusion Our study demonstrates that the efficiency of palbociclib and fulvestrant can be monitored by serial analyses of ctDNA before radiological evaluation and that early ctDNA variation is a prognostic factor of PFS.

Background Prognosis evaluation of advanced breast cancer and therapeutic strategy are mostly based on clinical features of advanced disease and molecular profiling of the primary tumor. Very few studies have evaluated the impact of metastatic subtyping during the initial metastatic event in a prospective study. The genomic landscape of metastatic breast cancer has mostly been described in very advanced, pretreated disease, limiting the findings transferability to clinical use. Methods We developed a multicenter, single-arm, prospective clinical trial in order to address these issues. Between November 2010 and September 2013, 123 eligible patients were included. Patients at the first, untreated metastatic event were eligible. All matched primary tumors and metastatic samples were centrally reviewed for pathological typing. Targeted and whole-exome sequencing was applied to matched pairs of frozen tissue. A multivariate overall survival analysis was performed (median follow-up 64 months). Results Per central review in 84 patients (out of 130), we show that luminal A breast tumors are more prone to subtype switching. By combining targeted sequencing of a 91 gene panel (n = 67) and whole-exome sequencing (n = 30), a slight excess of mutations is observed in the metastases. Luminal A breast cancer has the most heterogeneous mutational profile and the highest number of mutational signatures, when comparing primary tumor and the matched metastatic tissue. Tumors with a subtype change have more mutations that are private. The metastasis-specific mutation load is significantly higher in late than in de novo metastases. The most frequently mutated genes were TP53 and PIK3CA. The most frequent metastasis-specific druggable genes were PIK3CA, PTEN, KDR, ALK, CDKN2A, NOTCH4, POLE, SETD2, SF3B1, and TSC2. Long-term outcome is driven by a combination of tumor load and metastasis biology. Conclusions Profiling of the first, untreated, metastatic event of breast cancer reveals a profound heterogeneity mostly in luminal A tumors and in late metastases. Based on this profiling, we can derive information relevant to prognosis and therapeutic intervention, which support current guidelines recommending a biopsy at the first metastatic relapse. Trial registration The trial was registered at ClinicalTrials.gov (NCT01956552).

Contains fulltext : 245198.pdf (Publisher’s version ) (Closed access) High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland-Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from -0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the 'ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

Homologous Recombination DNA-repair deficiency (HRD) is a well-recognized marker of platinum-salt and PARP inhibitor chemotherapies in ovarian and breast cancers (BC). Causing high genomic instability, HRD is currently determined by BRCA1/2 sequencing or by genomic signatures, but its morphological manifestation is not well understood. Deep Learning (DL) is a powerful machine learning technique that has been recently shown to be capable of predicting genomic signatures from stained tissue slides. However, DL is known to be sensitive to dataset biases and lacks interpretability. Here, we present and evaluate a strategy to control for biases in retrospective cohorts. We train a deep-learning model to predict the HRD in a controlled cohort with unprecedented accuracy (AUC: 0.86) and we develop a new visualization technique that allows for automatic extraction of new morphological features related to HRD. We analyze in detail the extracted morphological patterns that open new hypotheses on the phenotypic impact of HRD.

High‐throughput DNA analyses in cancers allow the detection of molecular abnormalities that can guide patients' treatment. The collection of a tumour DNA is mainly performed on a biopsy, an invasive procedure for the patients. Another less invasive method consists in using a fine needle. We showed that fine‐needle biopsies are as suitable as a classical biopsy for DNA analysis., High‐throughput molecular profiling of solid tumours using core needle biopsies (CNB) allows the identification of actionable molecular alterations, with around 70% success rate. Although several studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to the sensitivity of the less invasive fine‐needle aspiration (FNA) compared to CNB to detect molecular alterations. We aimed to prospectively evaluate the potential of FNA to detect such alterations in various tumour types as compared to CNB in cancer patients included in the SHIVA02 trial. An in‐house amplicon‐based targeted sequencing panel (Illumina TSCA 99.3 kb panel covering 87 genes) was used to identify pathogenic variants and gene copy number variations (CNV) in concomitant CNB and FNA samples obtained from 61 patients enrolled in the SHIVA02 trial (NCT03084757). The main tumour types analysed were breast (38%), colon (15%), pancreas (11%), followed by cervix and stomach (7% each). We report 123 molecular alterations (85 variants, 23 amplifications and 15 homozygous deletions) among which 98 (80%) were concordant between CNB and FNA. The remaining discordances were mainly related to deletions status, yet undetected alterations were not exclusively specific to FNA. Comparative analysis of molecular alterations in CNB and FNA showed high concordance in terms of variants as well as CNVs identified. We conclude FNA could therefore be used in routine diagnostics workflow and clinical trials for tumour molecular profiling with the advantages of being minimally invasive and preserve tissue material needed for diagnostic, prognostic or theranostic purposes.

BackgroundAsiDNA, a first-in-class oligonucleotide-mimicking double-stranded DNA breaks, acts as a decoy agonist to DNA damage response in tumour cells. It also activates DNA-dependent protein kinase and poly (adenosine diphosphate [ADP]-ribose) polymerase enzymes that induce phosphorylation of H2AX and protein PARylation.MethodsThe aim of this Phase 1 study was to determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), safety and pharmacokinetics/pharmacodynamics of AsiDNA administered daily for 3 days in the first week then weekly thereafter. Twenty-two patients with advanced solid tumours were enrolled in 5 dose levels: 200, 400, 600, 900, and 1300 mg, using a 3 + 3 design.ResultsThe MTD was not reached. IV AsiDNA was safe. Two DLTs (grade 4 and grade 3 hepatic enzymes increased at 900 and 1300 mg), and two related SAE at 900 mg (grade 3 hypotension and grade 4 hepatic enzymes increased) were reported. AsiDNA PK increased proportionally with dose. A robust activation of DNA-PK by a significant posttreatment increase of γH2AX was evidenced in tumour biopsies.ConclusionThe dose of 600 mg was identified as the optimal dose for further clinical development.Clinical trial registrationClinical trial registration (NCT number): NCT03579628.

BackgroundEarly luminal breast cancer (BC) represents 70% of newly diagnosed BC cases. Among them, small (under 2 cm) BC without lymph node metastasis (classified as T1N0) have been rarely studied, as their prognosis is generally favorable. Nevertheless, up to 5% of luminal T1N0 BC patients relapse with distant metastases that ultimately prove fatal. The aim of our work was to identify the mechanisms involved in metastatic recurrence in these patients.MethodsOur study addresses the role that autonomous and non-autonomous tumor cell features play with regard to distant recurrence in early luminal BC patients. We created a cohort of T1N0 luminal BC patients (tumors between 0.5–2 cm without lymph node metastasis) with metastatic recurrence (“cases”) and corresponding “controls” (without relapse) matched 1:1 on main prognostic factors: age, grade, and proliferation. We deciphered different characteristics of cancer cells and their tumor micro-environment (TME) by deep analyses using immunohistochemistry. We performed in vitro functional assays and highlighted a new mechanism of cooperation between cancer cells and one particular subset of cancer-associated fibroblasts (CAF).ResultsWe found that specific TME features are indicative of relapse in early luminal BC. Indeed, quantitative histological analyses reveal that “cases” are characterized by significant accumulation of a particular CAF subset (CAF-S1) and decrease in CD4+T lymphocytes, without any other association with immune cells. In multivariate analysis, TME features, in particular CAF-S1 enrichment, remain significantly associated with recurrence, thereby demonstrating their clinical relevance. Finally, by performing functional analyses, we demonstrated that CAF-S1 pro-metastatic activity is mediated by the CDH11/osteoblast cadherin, consistent with bones being a major site of metastases in luminal BC patients.ConclusionsThis study shows that distant recurrence in T1N0 BC is strongly associated with the presence of CAF-S1 fibroblasts. Moreover, we identify CDH11 as a key player in CAF-S1-mediated pro-metastatic activity. This is independent of tumor cells and represents a new prognostic factor. These results could assist clinicians in identifying luminal BC patients with high risk of relapse. Targeted therapies against CAF-S1 using anti-FAP antibody or CDH11-targeting compounds might help in preventing relapse for such patients with activated stroma.

Prosigna® tests are performed in Institut Curie to guide adjuvant chemotherapy decision in early estrogen receptor (ER) positive, Human Epidermal growth factor Receptor (HER2) negative breast cancer (BC) patients, with an intermediate risk of recurrence. The objective of this study was to check the absence of an early warning signal for therapeutic decisions guided by the use of genomic tests in terms of the occurrence of events during the follow-up. Methods: Follow-up analysis were made on 2 populations: Real-life population: ER positive, HER2 negative BC patients who had a Prosigna® test in clinical routine, were prospectively registered and their data collected. Prosigna® test was indicated for patients with an intermediate risk of recurrence defined by our regional guidelines (1): grade 1 T1cN1, grade 1 T2N0-1, grade 2 T1cN0 with pejorative prognosis parameters (Ki67>20% or lympho-vascular emboli), grade 2 T2N0, grade 3 T1cN0. Decision impact (DI) study (2): a prospective multicenter study evaluating the decision impact of Prosigna® tests on adjuvant chemotherapy treatment. All consecutive T1-T2 NO-N1(mic) ER positive, HER2 negative BC patients were included. Survival was estimated by Kaplan-Meier methods and compared between patients who received or not chemotherapy based on the tests results with a Log-rank test (chemotherapy was administered for patients with a 10-years distant recurrence probability >=10%). Results are given in % with 95% intervalley confidence. Results: 647 patients were included (124 from DI study and 523 from clinical routine). Median age was 59 years-old (28-83). The median size of the tumor was 17 mm, (3-75), with a majority of grade 2 tumors (n=473, 73%) and 14% of lymph node involvement (n=90). Prosigna® tests results showed: 54% of luminal A.; respectively 30% (n=195), 40% (n=257) and 30% (n=195) of high, intermediate and low risk categories. The median 10-years distant recurrence probability was 10% (2-45%). The median follow-up of the patients was 18 months (0-52). We did not observe death in our study population, and we registered 10 recurrences (4 distant-DMFS- and 6 locoregional-LDFS). Disease free survival (DFS) at respectively 12 and 24 months were 99.8% (98.9-100) and 98.6% (96.5-99.4). Locoregional DFS at 12 and 24 months were 99.8% (98.9-100) and 99.2% (97.4-99.8) and distant DFS at 12 and 24 months were 100% and 99.4% (97.5-99.8). We did not observe survival differences between patients treated by chemotherapy and hormone therapy (CT) and patients treated by hormone therapy alone (No CT). DFS were respectively for CT and no CT groups 99.6% (97.4-99.9) and 99.5% (96.8-99.9, p=0.8) at 12 months, and 98.9% (95.1-99.7) and 99% (96-99.7, p=0.8) at 24 months. Distant and locoregional DFS were also not different between CT and No CT groups. Discussion: The first results of this study, mainly composed of clinical routine patients, are totally reassuring. We did not identify early warning signal in terms of survival with the use of Prosigna® test to guide adjuvant chemotherapy decisions in early BC patients at intermediate risk of recurrence. Moreover, our results are in accordance with the TAILORx (3) and MINDACT (4) randomized prospective studies survivals. References: 1. https://www.gustaveroussy.fr/sites/default/files/referentiel-cancer-du-sein-gustaveroussy-curie-2016-2017.pdf 2. Hequet D, et al. PLoS One. 2017 Oct 18;12(10):e0185753 3. Sparano JA, et al. N Engl J Med. 2018 Jul 12;379(2):111-121 4. Cardoso F, et al; N Engl J Med. 2016 Aug 25;375(8):717-29 Citation Format: Delphine Hequet, Louise Maumy, Celine Callens, David Gentien, Ivan Bieche, Paul Cottu, Jean-Marc Guinebretiere, Anne Vincent-Salomon, Florence Lerebours, Roman Rouzier. Outcomes results of Prosigna® test in early breast cancer patients: Experience of Institut Curie, France [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 P2-08-21.

The Prosigna® test has been performed in Institut Curie, France, since 2016 in clinical routine with a financial support of the health ministry dedicated to innovative procedures. The genomic platform at the Institut Curie performs the Prosigna® test for patients cared for at the Curie Hospital and in other institutions. Our main objective was to evaluate the impact of Prosigna® tests on adjuvant chemotherapy decisions. The second objective was to assess the costs associated with implementation of the test for patient management. Methods: Early breast cancer patients with estrogen receptor positive and human epidermal growth factor receptor 2-negative tumors, who had a Prosigna® test in clinical routine, were prospectively registered and observed. The Prosigna® test was indicated for a population candidate for a chemotherapy, with an intermediate risk of recurrence defined by our regional guidelines (1): grade 1 T1cN1, grade 1 T2N0-1, grade 2 T1cN0 with risk factors (Ki67>20% or lympho-vascular emboli), grade 2 T2N0, grade 3 T1cN0. The French Health Authority (FHA) has also recently defined the population who are candidates for genomic testing: T1c/T2 and N0/N1(mic) and grade 2 without risk factors (age Results: Prosigna® test was performed for 831 patients in clinical practice from 2016 and 2019 at Institut Curie. Due to missing data, analyses have been performed on 735 patients. Median age was 57 years-old (28-80), and median size 18 mm (3-80). We observed a majority of grade 2 tumors (n=572, 78%), and 21% of lymph node involvement. Median Ki67 was 20 (0-100). Prosigna® tests classified respectively 234 (32%), 295 (40%) and 206 (28%) in high, intermediate and low risk categories. In the all real-life population (n=735), chemotherapy was avoided for 308 (42%) patients after Prosigna® test results, and in 192 (44%) cases in the population defined by the FHA (n=433). From a financial point of view, both the real-life data and the population defined by FHA demonstrated cost savings,1 739 614€ (n=735 patients). and 1 068 671€(n=433), respectively. Conclusion: This large real-life study showed a 40% decrease in adjuvant chemotherapy recommendations after introduction of the Prosigna® test in a population of chemotherapy candidates. Moreover, this therapeutic de-escalation demonstrated an overall cost savings for the health system despite the added cost of the test. References: 1. https://www.gustaveroussy.fr/sites/default/files/referentiel-cancer-du-sein-gustaveroussy-curie-2016-2017.pdf 2. Baffert S, et al. The patient-breast cancer care pathway: how could it be optimized? BMC Cancer. 2015 May 12;15:394 3. Héquet D, et al. Direct medical and non-medical costs of a one-year care pathway for early operable breast cancer: results of a French multicenter prospective study. Plosone 2019, accepted, in press. Citation Format: Delphine Hequet, Guillaume Harrissart, Celine Callens, David Gentien, Ivan Bieche, Paul Cottu, Jean-Marc Guinebretiere, Anne Vincent-Salomon, Florence Lerebours, Roman Rouzier. Prosigna® test in clinical routine: Impact on adjuvant chemotherapy decision and medico-economic considerations in France [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 P2-14-10.

Histopathological assessment of ductal carcinoma in situ, a nonobligate precursor of invasive breast cancer, is characterized by considerable interobserver variability. Previously, post hoc dichotomization of multicategorical variables was used to determine the "ideal" cutoffs for dichotomous assessment. The present international multicenter study evaluated interobserver variability among 39 pathologists who performed upfront dichotomous evaluation of 149 consecutive ductal carcinomas in situ. All pathologists independently assessed nuclear atypia, necrosis, solid ductal carcinoma in situ architecture, calcifications, stromal architecture, and lobular cancerization in one digital slide per lesion. Stromal inflammation was assessed semiquantitatively. Tumor-infiltrating lymphocytes were quantified as percentages and dichotomously assessed with a cutoff at 50%. Krippendorff's alpha (KA), Cohen's kappa and intraclass correlation coefficient were calculated for the appropriate variables. Lobular cancerization (KA = 0.396), nuclear atypia (KA = 0.422), and stromal architecture (KA = 0.450) showed the highest interobserver variability. Stromal inflammation (KA = 0.564), dichotomously assessed tumor-infiltrating lymphocytes (KA = 0.520), and comedonecrosis (KA = 0.539) showed slightly lower interobserver disagreement. Solid ductal carcinoma in situ architecture (KA = 0.602) and calcifications (KA = 0.676) presented with the lowest interobserver variability. Semiquantitative assessment of stromal inflammation resulted in a slightly higher interobserver concordance than upfront dichotomous tumor-infiltrating lymphocytes assessment (KA = 0.564 versus KA = 0.520). High stromal inflammation corresponded best with dichotomously assessed tumor-infiltrating lymphocytes when the cutoff was set at 10% (kappa = 0.881). Nevertheless, a post hoc tumor-infiltrating lymphocytes cutoff set at 20% resulted in the highest interobserver agreement (KA = 0.669). Despite upfront dichotomous evaluation, the interobserver variability remains considerable and is at most acceptable, although it varies among the different histopathological features. Future studies should investigate its impact on ductal carcinoma in situ prognostication. Forthcoming machine learning algorithms may be useful to tackle this substantial diagnostic challenge. ispartof: MODERN PATHOLOGY vol:33 issue:3 pages:354-366 ispartof: location:United States status: published

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC. ispartof: NPJ BREAST CANCER vol:7 issue:1 ispartof: location:United States status: published

Simple Summary Recent findings have revealed the contribution of cancer-associated fibroblasts (CAF) in immune escape in breast cancer. Still, how to specifically target immunosuppressive CAF remains an unmet medical question. Here, we provide a promising therapeutic strategy by highlighting the role of CD73 in immunosuppressive CAF. By studying cohorts of breast cancer patients and performing functional assays, our study uncovers how CD73 contributes to immunosuppression by acting in a specific CAF subpopulation (referred to as CAF-S1) in breast cancer. In addition, we validate that using an anti-CD73 antibody significantly reduces CAF-S1-mediated immunosuppression, thereby highlighting a new interesting therapeutic strategy for breast cancer patients. Abstract Background: Cancer-associated fibroblasts (CAF) are heterogeneous with multiple functions in breast cancer. Recently, we identified a specific CAF subpopulation (referred to as CAF-S1), which promotes immunosuppression and immunotherapy resistance. Methods and Results: Here, by studying a large collection of human samples, we highlight the key function of CD73/NT5E in CAF-S1-mediated immunosuppression in breast cancer. We first reveal that CD73 protein level specifically accumulates in CAF-S1 in breast cancer patients. Interestingly, infiltration of regulatory T lymphocytes (Tregs) is significantly correlated with CD73 expression in stroma but not in epithelium, indicating that CD73 contributes to immunosuppression when expressed in CAF-S1 and not in tumor cells. By performing functional assays based on relevant systems using primary CAF-S1 isolated from patients, we demonstrate that CAF-S1 increase the content in both PD-1+ and CTLA-4+ Tregs. Importantly, the use of a blocking anti-CD73 antibody on CAF-S1 reduces CAF-S1-mediated immunosuppression by preventing expression of these immune checkpoints on Tregs. Conclusions: Our data support the potential clinical benefit of using both anti-CD73 and immune-checkpoint inhibitors in breast cancer patients for inhibiting CAF-S1-mediated immunosuppression and enhancing anti-tumor immune response.

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis and no dedicated therapy, therefore new tools to understand, detect, and treat these tumors are needed. Certain germline genes are re-expressed in tumors, and constitute the Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here, we designed a new approach to examine Cancer/ Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the 2 Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells but not the microenvironment, and that they are not expressed by normal breast progenitors, in other words their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Basal-like tumors expressing both genes have a poorer outcome than tumors expressing either gene alone or neither gene. Therefore, these findings suggest a potential synergistic effect between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worse outcomes.

Simple Summary The originality of this prospective study is to use radiation therapy in association with chemotherapy before surgery and permit patients to preserve their breasts or to undergo immediate reconstruction. This neoadjuvant strategy can therefore allow one-stage breast reconstructive surgery, the so-called “reverse technique”. Abstract Background: Neoadjuvant concurrent radiochemotherapy makes it possible to increase the breast conservation rate. This study reports the long term outcome of this treatment. Methods: From 2001 to 2003, 59 women with T2–3 N0–2 M0 invasive breast cancer (BC) not amenable to upfront breast conserving treatment (BCS) were included in this prospective, non-randomized phase II study. Chemotherapy (CT) consisted of four cycles of continuous 5-FU infusion and Vinorelbine. Starting concurrently with the second CT cycle, normofractionated RT was delivered to the breast and LN. Breast surgery was then performed. Results: Median follow-up (FU) was 13 years [3–18]. BCS was performed in 41 (69%) patients and mastectomy in 18 patients, with pathological complete response rate of 27%. Overall and distant-disease free survivals rates at 13 years were 70.9% [95% CI 59.6–84.2] and 71.5% [95% CI 60.5–84.5] respectively. Loco regional and local controls rates were 83.4% [95% CI 73.2–95.0] and 92.1% [95% CI 83.7–100], respectively. Late toxicity (CTCAE-V3) was assessed in 51 patients (86%) with a median follow-up of 13 years. Fifteen presented grade 2 fibrosis (29.4%), 8 (15.7%) had telangiectasia, and 1 had radiodermatitis. Conclusions: This combined treatment provided high long-term local control rates with limited side-effects.

Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer. TILs can be easily assessed on hematoxylin and eosin stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Since TILs and PD-L1 are parts of an immunological spectrum in breast cancer, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immune-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with breast cancer. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in breast cancer. This article is protected by copyright. All rights reserved., info:eu-repo/semantics/published

International audience; Purpose of review. The aim of this study was to highlight the diagnostic and management challenges of primary vitreoretinal lymphoma (PVRL) through a review of the literature and a European survey on real-life practices for PVRL.Recent findings. The care of PVRL patients is heterogeneous between specialists and countries. Upfront systemic treatment based on highdose methotrexate chemotherapy, with or without local treatment, might reduce or delay the risk of brain relapse. Ibrutinib, lenalidomide with or without rituximab, and temozolomide are effective for patients with relapsed/refractory PVRL and should be tested as first-line treatments.Summary. The prognosis of PVRL remains dismal. No firm conclusion regarding optimal treatment can yet be drawn. The risk of brain relapse remains high. Diagnostic procedures and assessment of therapeutic responses need to be homogenized. Collaboration between specialists involved in PVRL and multicentric prospective therapeutic studies are strongly needed. The recommendations of the French group for primary oculocerebral lymphoma (LOC network) are provided, as a basis for further European collaborative work.

Malignant adenomyoepithelioma (AME) of the breast is an exceptionally rare form of breast cancer, with a significant metastatic potential. Chemotherapy has been used in the management of advanced AME patients, however the majority of treatments are not effective. Recent studies report recurrent mutations in the HRAS Q61 hotspot in small series of AMEs, but there are no preclinical or clinical data showing H-Ras protein as a potential therapeutic target in malignant AMEs. We performed targeted sequencing of tumours’ samples from new series of 13 AMEs, including 9 benign and 4 malignant forms. Samples from the breast tumour and the matched axillary metastasis of one malignant HRAS mutated AME were engrafted and two patient-derived xenografts (PDX) were established that reproduced the typical AME morphology. The metastasis-derived PDX was treated in vivo by different chemotherapies and a combination of MEK and BRAF inhibitors (trametinib and dabrafenib). All malignant AMEs presented a recurrent mutation in the HRAS G13R or G12S hotspot. Mutation of PIK3CA were found in both benign and malignant AMEs, while AKT1 mutations were restricted to benign AMEs. Treatment of the PDX by the MEK inhibitor trametinib, resulted in a marked anti-tumor activity, in contrast to the BRAF inhibitor and the different chemotherapies that were ineffective. Overall, these findings further expand on the genetic features of AMEs and suggest that patients carrying advanced HRAS-mutated AMEs could potentially be treated with MEK inhibitors.

The last international guidelines on HER2 determination in breast cancer have been updated in 2018 by the American Society of Clinical Oncology and College of American Pathologists, on the basis of a twenty-year practice and results of numerous clinical trials. Moreover, the emerging HER2-low concept for 1+ and 2+ non amplified breast cancers lead to refine French practices for HER2 status assessment. The GEFPICS group, composed of expert pathologists, herein presents the latest French recommendations for HER2 status evaluation in breast cancer, taking into account the ASCO/CAP guidelines and introducing the HER2-low concept. In the era of personalized medicine, HER2 status assessment remains one of the most important biomarkers in breast cancer and its quality guaranties the optimal patients' care. French pathologists' commitment in theranostic biomarker quality is more than ever required to provide the most efficient cares in oncology.

Objective Tumors harboring a POLE pathogenic variant, associated with high tumor mutational burden, are good candidates for immunotherapy. However, POLE pathogenic variants are not currently screened in routine clinical practice. Can these tumors be identified by means of an already available test? Methods We describe seven tumors harboring a POLE pathogenic variant, among eight patients with tumors harboring multiple BRCA1/2 variants (from 4 to 20). All patients were managed at Institut Curie, Paris. Five patients were selected because of unexpected tumor BRCA testing results with multiple variants and another three patients were selected because of a POLE pathogenic variant detected by large tumor testing. We looked for other tumor variants by Next-Generation Sequencing in tumors harboring multiple BRCA1/2 variants, and for multiple BRCA1/2 variants in tumors harboring a POLE pathogenic variant. Results Four of the five tumors selected because of multiple BRCA1/2 variants exhibited a POLE pathogenic variant, and all three tumors selected for POLE pathogenic variants exhibited multiple BRCA1/2 variants. Conclusions Tumor BRCA testing could be a way to detect tumors harboring a highly mutagenic POLE pathogenic variant.

Simple Summary Invasive lobular breast cancer (ILC) is a special type of breast cancer (BC) that was first described in 1941. The diagnosis of ILC is made by microscopy of tumor specimens, which reveals a distinct morphology. This review recapitulates the developments in the microscopic assessment of ILC from 1941 until today. We discuss different concepts of ILC, provide an overview on ILC variants, and highlight advances which have contributed to a better understanding of ILC as a special histologic spectrum of tumors. Abstract Invasive lobular breast cancer (ILC) is the most common special histological type of breast cancer (BC). This review recapitulates developments in the histomorphologic assessment of ILC from its beginnings with the seminal work of Foote and Stewart, which was published in 1941, until today. We discuss different concepts of ILC and their implications. These concepts include (i) BC arising from mammary lobules, (ii) BC growing in dissociated cells and single files, and (iii) BC defined as a morpho-molecular spectrum of tumors with distinct histological and molecular characteristics related to impaired cell adhesion. This review also provides a comprehensive overview of ILC variants, their histomorphology, and differential diagnosis. Furthermore, this review highlights recent advances which have contributed to a better understanding of the histomorphology of ILC, such as the role of the basal lamina component laminin, the molecular specificities of triple-negative ILC, and E-cadherin to P-cadherin expression switching as the molecular determinant of tubular elements in CDH1-deficient ILC. Last but not least, we provide a detailed account of the tumor microenvironment in ILC, including tumor infiltrating lymphocyte (TIL) levels, which are comparatively low in ILC compared to other BCs, but correlate with clinical outcome. The distinct histomorphology of ILC clearly reflects a special tumor biology. In the clinic, special treatment strategies have been established for triple-negative, HER2-positive, and ER-positive BC. Treatment specialization for patients diagnosed with ILC is just in its beginnings. Accordingly, ILC deserves greater attention as a special tumor entity in BC diagnostics, patient care, and cancer research.

Neoadjuvant therapy is an increasing treatment option in the management of breast cancer. The tumor response to neoadjuvant therapy, especially the pathological complete response, is a validated endpoint frequently used in clinical trials. However, there is still a lack of standardization for the surgical specimen management in the neoadjuvant setting. This leads to heterogeneity in the specimen handling and might lead to significant bias for the prognostic assessment of patients or in clinical trials. The GEFPICS group, composed of expert breast cancer pathologists, herein presents guidelines for the management of breast and axillary specimen before treatment (management of biopsy, items of the pathological report) and after neoadjuvant therapy (specimen handling, histological assessment of response, items of the pathological report and response grading systems).

A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting. ispartof: Translational Oncology vol:12 issue:12 pages:1557-1565 ispartof: location:United States status: Published online

Secretory carcinoma is a special-type breast carcinoma underpinned by a recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion. Immunohistochemistry (IHC) using a pan-TRK antibody has been recently shown to help identify NTRK rearrangements in other tumor types. The purpose of this study was to assess the diagnostic utility of pan-TRK IHC in secretory carcinoma of the breast. Pan-TRK IHC was performed using a rabbit monoclonal antibody on whole sections of 24 breast secretory carcinomas and tissue microarray sections of other breast carcinoma types (n=203) and histologic mimics (n=15). Cases were assessed for staining intensity and localization. The 24 patients with secretory carcinoma had a median age of 44 years and a median tumor size of 1.0 cm. ETV6 fluorescence in situ hybridization was positive in all cases tested (n=20). Twenty-three cases (95.8%) showed staining with pan-TRK, which was exclusively nuclear in 19, primarily nuclear with weak cytoplasmic staining in 3, and primarily cytoplasmic with focal nuclear staining in 1. The nuclear staining was diffuse in 17 and at least focally strong in 17. The only pan-TRK negative case was a core biopsy with limited tumor. Among the 203 nonsecretory carcinomas, 21 (10.3%) showed focal, weak nuclear staining in

Purpose: Human papillomavirus (HPV) is found in 90% of squamous cell carcinomas of the anal canal (SCCA). We investigated the clinical validity of HPV circulating tumor DNA (ctDNA) detection in patients enrolled in the Epitopes-HPV02 trial that demonstrated the efficacy of docetaxel, cisplatin, and 5-FU as first-line chemotherapy in advanced SCCA. Experimental Design: According to the protocol, serum samples were collected before chemotherapy and on completion of chemotherapy. HPV16 ctDNA was quantified by droplet digital PCR (ddPCR) and correlated with prospectively registered patient characteristics and outcomes. A landmark was set at the time of chemotherapy completion for postchemotherapy progression-free survival (PFS) analyses. Results: Among 57 patients with HPV16-related advanced SCCA, HPV ctDNA was detected in 91.1% (95% confidence interval, 81.1–96.2) of baseline samples. Baseline HPV ctDNA levels were not associated with any patient characteristics; baseline ctDNA level below the cutoff obtained by AUC (area under the curve) was associated with a longer PFS (HR = 2.1; P = 0.04). Among the 36 patients who completed 5 months of chemotherapy, residual HPV ctDNA was detected after chemotherapy in 38.9% of patients. Residual HPV ctDNA detected at chemotherapy completion was associated with shorter postchemotherapy PFS (median PFS 3.4 months vs. not reached; HR = 5.5; P < 0.001) and a reduction of 1-year overall survival rate (OR = 7.0; P = 0.02). Conclusions: This prospective study in advanced SCCA demonstrated a significant prognostic impact of HPV ctDNA level before first-line chemotherapy and HPV ctDNA negativity after chemotherapy completion. With a limited cost and short turnaround, this assay is a promising tool to optimize the therapeutic management of SCCA. See related commentary by Morris, p. 2030

Introduction Li-Fraumeni syndrome (LFS) resulting from monoallelic germline TP53 gene mutation is a rare hereditary cancer predisposition. Breast cancer (BC) is the most common cancer among women with TP53 germline mutation with a risk ranging from 49% to 85% by the age of 60 years old. Most of these cancers are early onset. Few patients' cases have been reported so far in the literature. Our aim was to describe the medical history of a cohort of LFS women diagnosed with BC recruited from a single institution. The characteristics combined were genetic alteration diagnosis, tumor characteristics, treatment, outcome, and LFS associated cancers. Methods We retrospectively identified breast cancer patients with TP53 germline mutation from the Institut Curie (Paris, France) database and described their cancer characteristics and medical history. Results From 1989 to 2015, 25 patients affected with BC (31 tumors) and TP53 germline mutation carrier were identified, with a median follow up of 6.5 years. Median age at BC diagnosis was 30.5 years. All patients were women. 33% had a previously identified TP53 mutation in their family. 70% of them had BC as their first cancer event. 60% of the patients presented with another LFS associated cancer or non-related cancers: osteosarcoma (22%), glioblastoma (18%), pulmonary carcinoma (13%), gastric linitis plastica (9%), malignant hemopathy (9%), soft tissue sarcoma (9%), adrenocortical carcinoma (4%), ovarian cystadenocarcinoma (4%), renal tumor (4%), choroid plexus carcinoma (4%). 92% of the breast tumors were ductal carcinoma (28% DCIS and 64% IDC), 7% were sarcoma (1 phyllodesarcoma, 1 pleiomorphic liposarcoma); there were no lobular carcinoma. Among the IDC, 50% were HER2 positive, 72% were hormone-receptor positive. Most patients had a mastectomy (64%), and most of them received radiation (55%). However, when TP53 mutation had been identified prior to the treatment, none of the patients received radiotherapy (5 patients). Most patients received chemotherapy (70%) (37% in neoadjuvant setting, 33% in adjuvant setting, 25% for metastatic setting). 40% of the patients received hormone therapy (37% as adjuvant treatment, 7% for metastatic disease) Most of the patients did not relapse from BC (75%). Overall, only 17% of the patients had metastatic BC. To date, 12 patients of our series have died (48%), 6 patients (24%) from other LFS-associated cancers and 4 patients from BC (16%). Conclusion To the best of our knowledge, this descriptive series is the largest study of tumor characteristics and medical history of LFS-women with BC, the most frequent cancer among women with TP53 germline mutation. It confirms the higher HER2 overexpression rate observed in LFS-patients BC, as suggested in the literature and showed a high rate of DCIS at initial presentation. Most of the patients developed other LFS-associated cancers. In depth molecular analysis of these BC will be performed in order to gain insight into their biological specificities and to adapt the therapeutic management of this poor prognosis syndrome. Citation Format: Tran M, Loirat D, Colas C, Bozec L, Laurence V, Lerebours F, Cabel L, Bidard F-C, Stoppa-Lyonnet D, Vincent-Salomon A, Gauthier-Villars M, Lavigne M, De Pauw A. Breast cancer characteristics and outcomes in patients with TP53 germline mutation [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 P1-09-07.

Background The EndoPredict (EP) test has been developed and validated for assessing recurrence risk in patients with estrogen receptor (ER)-positive HER2-negative breast cancer (BC). This test is based on the expression of 12 genes (molecular score), combined with clinicopathological criteria (i.e. tumor size and nodal status) (EPclin risk score). For node-positive patients, the weight given to the node status in the EPclin score is similar when considering pN1mi (]0.2-2mm]) or pN1 (>2mm, 1-3 positive lymph nodes). Our aim was to characterize the EPclin classification of the pN1mi subgroup in the French national registry for molecular signatures in ER+ BC. Methods Since April 2016, nine French laboratories using EP test in clinical practice prospectively implement a national registry for molecular prognostic signatures in ER+ BC. We analyzed the pN1mi subgroup with regards to their clinico-pathological characteristics, molecular EP score and EPclin risk score. Using the definition formula of the EPclin score [EPclin=0.35t + 0.64n + 0.28EP, with n=1 for pN0, 2 for pN1mi/pN1, 3 for pN2, 4 for pN3], we could calculate a hypothetical EPclin score if the pN1mi had been considered as pN0 [n factor=1]. Results By the end of 2017, the database included 1246 EP tests performed in routine practice, including 67 (5.4%) pN1mi. The pN1mi BC were ER+ HER2- (67/67, 100%), invasive carcinoma of no special type in 52/67 (78%), with a median tumor size of 18 mm (range 7-45; pT1c in 40/67). Among these, 22 were classified as EPclin low and 45 as EPclin high, with a median EP score of 6 (range 2-14), a median EPclin score of 3.70 (range 3-6), and a median relapse risk at 10-year of 15%(range 5-73). Most interestingly, 23/67 (34%; i.e. 1.8% of all EP tests performed) pN1mi BC displayed an EPclin score comprised between 3.4 and 4, just above the cut-off value of 3.3: these cases (and only these cases) would have been classified in a different risk category (i.e. EPclin low risk) if the node status would have been considered as negative. Conclusion With regards to EndoPredict test in pN1mi BC - and the persistent debate as to whether they should bear the same weight as node-positive (pN1) or negative (pN0) tumors - categorization in the EPclin class is not likely to be impacted by the node factor weight in the vast majority (>65%) of the pN1mi cases and >98% of all patients tested with EP. Only cases with an EPclin between 3.4 and 4 might be impacted. Citation Format: LaCroix-Triki M, Arnould L, MacGrogan G, Penault-Llorca F, Haudebourg J, Tas P, Garnier A, Vincent-Salomon A, Miquel C, Lehmann-Che J, Callens C, Quillien V, Cayre A, Lamy P-J, Rouleau E, De Cremoux P. EndoPredict prognostic signature in pN1mi, estrogen receptor-positive breast cancer: analysis of the French national registry for molecular signatures [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 P4-08-23.

Background: Therapeutic options at diagnosis for metastatic breast cancers (MBC) differ largely according to cancer phenotype (namely, hormone receptor (HR) and HER2 status). Reported discordance rates between primary tumor and metastasis vary widely in literature, with a median of 18% for estrogen receptor, 31% for progesterone receptor and 10% for HER2. The present study aimed to compare phenotypic profiles between primary and MBC in the real-life setting. Patients (pts) and methods: Epidemio-Strategy and Medical Economics (ESME)MBC data platform (NCT03275311) is a French national, multicenter, observational cohort using clinical trials' methodology for data capture, monitoring and quality controls. At the time of analysis, it comprised data of 16703 consecutive newly diagnosed MBC pts (1/01/08-31/12/14) treated in 18 French comprehensive cancer centres. The primary endpoint was the discordance rate of HR and HER2 status between primary tumor and MBC (biopsy of metastatic site done within 6 months of MBC diagnosis). Only patients with both histological reports available were considered. Potential factors associated with phenotype discordance were assessed in a multivariate logistic regression. Results: 2933 out of 16703 (17.6%) had a biopsy in the first 6 months of metastatic disease. HR and/or HER2 status was available in 1677 pts. The discordance rate between primary and matched MBC was 14.2% (222/1566) for HR: loss of expression in 72.5%, gain in 27.5%. For HER2, the discordance rate was 7.8% (84/1076): 45.2% of losses and 54.8% of gains of expression. The primary HR+/HER2+ subgroup had the highest rate of changes: 53% (49/92) with either a loss of HR (43%), loss of HER2 (43%) or a loss of both (14%). 18% (33/181) of primary triple-negative breast cancer (TNBC) had a phenotypic change with a majority of HR gain (79%). In multivariate analysis, administration of adjuvant chemotherapy +/- targeted therapy was the sole independent predictor of HR status modification (OR: 1.73, 95%CI 1.27-2.36, p=0.001). The presence of a mixed histology was the only predictor of HER2 discordance (OR =2.57, 95%CI 1.19-5.55, p=0.016). Patient characteristics Total population (n=16703)Pts with primary and MBC phenotype available (n=1677)Age at metastatic diagnosis Median (range)61 (19-99)60 (24-93)De novo MBC4507 (27.1%)221 (13.2%)Number of metastatic sites Median (range)1 (1-9)2 (1-7)MBC sites Brain1200 (7.2%)138 (8.2%)Visceral7755 (46.4%)928 (55.3%)Non-visceral7748 (46.4%)611 (36.4%)Phenotypic profileN = 2933N=1677TNBC356 (18.5%)272 (19.3%)HR+/HER2-1251 (65.0%)917 (65.2%)HR-/HER2+150 (7.8%)105 (7.5%)HR+/HER2+168 (8.7%)112 (8%)Missing1008271Metastatic site samplingN=2933N=1677Bone692 (24.2%)419 (25.5%)Liver514 (18.0%)355 (21.6%)Skin379 (13.3%)203 (12.4%)Node306 (10.7%)169 (10.3%)Lung258 (9.0%)168 (10.2%)Pleura283 (9.9%)121 (7.4%)CNS/CSF*132 (4.6%)42 (2.6%)Other or multiple296 (10.3%)165 (10.0%)Missing7335* CNS= central nervous system, CSF=cerebro-spinal fluid Conclusion: Biopsy and phenotype re-evaluation of MBC early in the disease course has a confirmed potential significant therapeutic impact in this large scale real life setting and should be proposed as often as possible. Citation Format: Lefevre S, Lusque A, Joyon N, Arnould L, Penault-Llorca F, MacGrogan G, Treilleux I, Vincent-Salomon A, Haudebourg J, Maran-Gonzalez A, Charafe-Jauffret E, Courtinard C, Franchet C, Verriele V, Brain E, Tas P, Delaloge S, Filleron T, LaCroix-Triki M. Phenotypic discordance between primary and metastatic breast cancer (MBC) in a large scale real-life multicentre French cohort [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 P5-12-05.

Background HER2 breast cancer status determines patients' eligibility for targeted therapy. HER2 level of amplification is associated with a better response to anti-HER2 therapy (Arnould CCR 2007, Singer CCR 2017). Benefit of anti-HER2 therapy for equivocal cases remains debated. Objectives We aimed to better characterize HER2-equivocal breast cancers by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) according to 2013 ASCO/CAP guidelines using PAM50 gene expression-based molecular subtyping. We then investigated genome-wide copy number alterations of HER2-equivocal cases, assessing agreement between genomic alterations of the chromosome (chr) 17 and molecular subtypes. Methods PAM50 (nCounter assay; Nanostring) was performed on RNA from formalin-fixed paraffin-embedded samples of 40 HER2-equivocal cases. These cases were subsequently analyzed by Agilent 60-mer oligonucleotide microarrays for array-based comparative genomic hybridization (aCGH). Results The 40 HER2-equivocal cases were classified as Luminal B in 16 cases (40%), HER2-Enriched in 14 cases (35%), Luminal A in 9 cases (22.5%) and Basal-like in 1 case (2.5%) using PAM50. By IHC, 34 cases (85%) were ER+, 24 (60%) were also PgR+, 26 (65%) were grade III and 33 (82.5%) showed a high Ki67 > 20%. Using aCGH, 10 cases (25%) presented chr 17q large copy number gain, 10 (25%) showed segmental copy number gains including HER2, 9 (22.5%) showed HER2 amplification, one (2.5%) showed a large copy number loss and 10 cases (25%) didn't show any copy number alteration of the chr 17. Out of the 14 PAM50 HER2-Enriched cases, only 5 (35.7%) showed HER2 genomic amplification (Table 1). Four HER2 amplified cases at the genomic level were classified as Luminal B (3 cases, Ki67 > 20%, ER+, PgR- by IHC) or Luminal A (1 case, Ki6710% by IHC) using PAM50, although these luminal B tumors presented strong correlation with the HER2-Enriched centroid. In total, 13 cases (32.5%) were discordant between molecular classification and genomic alteration status of the chr 17. Among patients with early stage HER2-equivocal breast cancers (n=37), 2 received neo-adjuvant chemotherapy (5.4%), 25 received adjuvant chemotherapy (67.6%) and 2 received adjuvant trastuzumab (5.4%). With a median follow up of 5.8 years (3.8-6.9), one controlateral recurrence (2.7%), four metastatic recurrences (10.8%) and three deaths were observed (8.1%). Conclusion Using PAM50, the majority of HER2-equivocal cases were classified as Luminal tumors. At the genomic level, HER2-equivocal cases harbored mostly chr 17 segmental or large copy number gains. These results emphasized the need of HER2 status genomic determination. In line with the new ASCO 2018 recommendations intending to decrease the number of cases considered as HER2 equivocal, we showed that these tumors were mainly HER2 not amplified, ER positive and grade 3. There is no evidence of benefit of anti-HER2 therapy in these cases. Table 1Genomic alterations of chromosome 17Basal-likeHER2-EnrichedLuminal ALuminal BTotalHER2 amplified05139Large copy number gain013610Segmental copy number gain153110No alteration032510Large copy number loss00011Total11491640 Citation Format: Borcoman E, Ngo C, Rapinat A, Simaga F, Mariani O, Fuhrmann L, Jeannot E, Laé M, Pierga J-Y, Gentien D, Pierron G, Morel P, Brauer HA, Vincent-Salomon A. PAM50 and CGH-array genomic characterization of HER2-equivocal breast cancers defined by the ASCO/CAP2013 recommendations and response to treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-25.

Background Many countries restrict patient material exchange to central diagnostic laboratories abroad, limiting access to assays like MammaPrint® (MP) and BluePrint® (BP). Both assays are microarray-based, with MP being prognostic for distant recurrence and BP for molecular subtyping of breast cancer (Luminal-, HER2-, and Basal-type). To increase accessibility, decentralization is required with Next Generation Sequencing (NGS) being the preferred testing platform given that most diagnostic laboratories have the technology in place. The aim of this beta testing study is to validate a previously developed and centrally validated MP and BP NGS kit for RNA samples in two large tertiary academic hospitals in Europe. Patients and Methods Patients with early breast cancer diagnosed at the Multidisciplinary Breast Center at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Patients with bilateral breast cancer or presenting with more than 3 positive lymph nodes were excluded. Only patients with invasive ductal and invasive lobular carcinoma were included. Twenty tissue sections were cut from formalin-fixed, paraffin-embedded (FFPE) blocks; 10 tissue sections were analyzed at the local site using the MP and BP NGS kit, and 10 tissue sections were analyzed at Agendia using the same kit and procedure, as well as with the golden standard method (gene expression microarrays). Targeted RNA sequencing of the 70 MP and 80 BP signature genes was performed on Illumina MiSeq instruments. The raw NGS data generated at the local test sites was sent through a secure file transfer protocol server to Agendia for interpretation and comparison with microarray and NGS performed in the Agendia laboratories. We aimed for a minimum concordance rate between MP and BP outcome of 90% between each local site and Agendia's centralized site. Results In this study, 116 early breast cancer patients were included (73 from University Hospitals Leuven and 43 from Curie Institute). Out of these patients, 52% were MP Low Risk and 48% MP High Risk according to microarray. The patients had a BP luminal, HER2 or basal subtype in respectively 83%, 9% and 8%. Concordance between MP microarray obtained from Agendia and MP NGS obtained from the local sites was 91.4%. Concordance between MP High and Low Risk classification between NGS Leuven versus NGS Agendia was 92.1% and between NGS Curie versus NGS Agendia 95.3%. For BP subtype outcomes, the results from microarray versus NGS for all patients combined from both local sites gave a 98.3% concordance and NGS Agendia versus NGS from each local site gave a 100% concordance. Conclusion The MP and BP NGS kit was successfully validated in a decentralized setting, showing high concordance between results obtained at three different sites. There was a clear benefit of having well-trained NGS experienced diagnostic technical teams. The MP and BP NGS kit the first FFPE targeted RNA sequencing based multigene signature for breast cancer care, will provide a high and equal standard of MP and BP gene expression testing for breast cancer in a decentralized setting. Citation Format: Slembrouck L, Laurent C, Delahaye LJ, Mittempergher L, Vanden Bempt I, Vander Borght S, Darrigues L, Vliegen L, Sintubin P, Raynal V, Bohec M, Reyes C, Rapinat A, Helsmoortel C, Jongen L, Hoste G, Neven P, Wildiers H, Smeets A, Nevelsteen I, Punie K, Van Nieuwenhuysen E, Han S, Salomon AV, Faron EL, Cynober T, Witteveen AT, Neijenhuis S, Glas AM, Reyal F, Floris G. Decentralized beta testing of MammaPrint and BluePrint NGS kit at University Hospitals Leuven and Curie Institute Paris [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 P4-08-25.

Background The role of chemotherapy in early luminal breast cancer remains challenged. The NEOPAL trial (NCT 02400567; Cottu et al, ESMO 2017 LBA09) compared sequential chemotherapy (CT) and letrozole-palbociclib (LP) as neoadjuvant treatment in PAM50 defined high-risk luminal breast cancer patients, showing that LP might be as efficient as CT with regard to breast conserving surgery and pathological response. We report here extended exploratory pathological results, focusing on tumor infiltrating lymphocytes (TILs), proliferative response and preoperative endocrine prognostic index (PEPI) scores. Material and Methods Tumor blocks from baseline biopsy and surgical specimens were available for centralized review from the 106 randomized patients (53 in each arm). TILs quantification, KI67 staining and counting, and ER quantification were performed according to standard methods. Residual proliferative cancer burden (RPCB) and PEPI scores were computed according to published algorithms. Wilcoxon rank sum test and Mann Whitney test were used to compare paired and unpaired data. The chi-square and Fisher exact tests were used for categorical variables. Results Overall, median TILs count did not differ between LP and CT patients, both at baseline (p=0.37) and at the end of treatment (p=0.42). Median TILs count climbed from 5% (0-60) to 10% (1-60) in the LP arm (p=0.0026) and from 2% (0-30) to 10% (0-60) in the CT arm (p=0.0023). Median Ki67 dropped sharply in both arms, from 30% (1-80) to 1% (0-30) in the LP arm (p=1.10e-8) and from 30% (2-80) to 5% (0-30) in the CT arm (p=3.10e-9). Decrease in the Ki67 geometric mean was as sharp. Of note, while baseline Ki67 was similar in both arms (p=0.315), decrease in the LP arm was significantly more profound than in the CT arm (p=0.00075). Pathological response according to RPCB were as follows, in the LP and CT arm, respectively: class 0: 9.6%/10.2%; class I: 84.6%/73.5%; class II: 5.8%/16.3%. The relapse free survival PEPI scores were as follow in the LP and CT arm, respectively: class I: 13.5%/16.3%; class II: 59.6%/46.9%; class III: 28.9%/36.8% (p=0.504). Breast cancer specific survival PEPI scores were as follow in the LP and CT arm, respectively: class I: 18.9%/8.2%; class II: 54.7%/40.8%; class III: 26.4%/51%. These results were significantly better in the LP arm (p=0.027). There was no correlation between final TILs quantification and the RPCB or PEPI scores. Conclusions In this prospective multicenter study with centralized pathological review, neoadjuvant letrozole-palbociclib combination generates impressive proliferative and endocrine specific response features. It compared well with chemotherapy. The LP combination also significantly increased lymphocytic infiltration. Its clinical significance and utility remain to be elucidated, but it potentially adds new prognostic and theranostic information. Citation Format: Vincent-Salomon A, Mathieu M-C, Bataillon G, Arnould L, Verrièle V, Ghnassia J-P, Haudebourg J, Penault-Llorca F, Lefebvre C, Maran-Gonzalez A, Guinebretière J-M, Duprez R, Berghian A, Blanc-Fournier C, Calès V, Galant C, Delrée P, Lemonnier J, Delaloge S, Cottu PH. TILs variations, proliferative response and PEPI scores in patients with luminal breast cancer receiving neoadjuvant letrozole-palbociclib or chemotherapy: An extended analysis of the NEOPAL trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-15-02.

Purpose: High levels of tumor-infiltrating lymphocytes (TILs) before neoadjuvant chemotherapy (NAC) are associated with higher pathological complete response (pCR) rates, and better survival in TNBC and HER2-positive breast cancers (BCs). We investigated the value of changes in TIL levels and final TIL levels after treatment, by evaluating lymphocyte infiltration before and after NAC in a real-life BC cohort. Patients and methods: We assessed stromal TIL levels in 716 pre- and post-treatment matched paired specimens, according to the guidelines of the international TIL working group. Results: Pre-NAC TIL levels were higher in tumors for which pCR was achieved than in cases of residual disease (33.9% versus 20.3%, p=0.001), in luminal tumors and TNBCs, but not in HER2-positive BCs, (pInteraction =0.001). The association between pre-NAC TIL levels and pCR was non-linear in TNBCs (p=0.005). Mean TIL levels decreased during NAC (pre-NAC TILs: 24.1% versus post-NAC TILs: 13.0%, p Conclusion: The associations of pre, post-NAC TIL levels with response to treatment and DFS differ between BC subtypes and may deviate from linearity. The characterization of immune subpopulations may improve our understanding of the complex interactions between pre- or post-NAC setting, BC subtype, response to treatment and prognosis. Citation Format: Hamy A-S, Bonsang-Kitzis H, De Croze D, Laas E, Darrigues L, Topciu L, Menet E, Vincent-Salomon A, Lerebours F, Pierga J-Y, Brain E, Feron J-G, Benchimol G, Lam G-T, Laé M, Reyal F. Interaction between molecular subtype and stromal immune infiltration dynamics in breast cancer patients treated with neoadjuvant chemotherapy [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 P3-11-01.

Background: Invasive lobular breast cancer (ILC) represents the second most common histology of breast cancer, accounts for 10-15% of all invasive cases and generally expresses the estrogen receptor (ER, coded by the ESR1 gene). Little is known about the genomic alterations associated with tumor progression and endocrine resistance in ILC. Here, we therefore molecularly characterized a unique series of matched primary and metastatic ILC. Patients and methods: We retrospectively identified 129 metastatic ER-positive ILC patients from 6 institutions. Following central pathology review and available DNA from the primary tumor (P), the metastasi(e)s (M), as well as from normal tissue, 80 patients (279 samples) were eligible for this study. All but 6 patients (7.5%) received endocrine treatment before metastatic sampling. Low pass whole genome and targeted gene screen (N=20 genes) sequencing was conducted to detect copy number aberrations (CNAs) and mutations associated with ILC metastatic progression respectively. ESR1 mutations were further assessed using droplet digital PCR (ddPCR). Publicly available data from IJB (n=413 ILC Ps), TCGA (n=172 ILC Ps), and MSKCC-IMPACT (n=116 ILC Ms) were used to compare and validate the frequencies of the detected alterations in ILC. Stromal tumor infiltrating lymphocytes (TILs) were assessed by two experienced pathologists. Results: The overall matched CNA comparison revealed a significant positive association between relapse-free survival and the P/M genomic distance defined by the number of CNAs private to P or M (r2= 0.52, p Regarding the immune infiltration, higher TILs in Ps were significantly associated with younger age at diagnosis, high grade tumors, and with mixed non-classic and trabecular histology. A paired analysis revealed no significant difference in TIL levels between P and M. TIL levels in the P or M were not associated with survival. Conclusion: This is to our knowledge the largest metastatic ILC series in which matched P and M samples were interrogated, revealing several genomic alterations, some of which potentially targetable, driving disease progression and endocrine resistance. Citation Format: Desmedt C, Richard F, Majjaj S, Pingitore J, Brown D, Rothé F, Marchio C, Clatot F, Mariani O, Boeckx B, Rouas G, Bertucci F, Galant C, Van den Eynden G, Salgado R, Lambrechts D, Vincent-Salomon A, Piccart M, Pruneri G, Larsimont D, Sotiriou C. Unraveling lobular breast cancer progression and endocrine resistance mechanisms through genomic and immune characterization of matched primary and metastatic samples [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 GS1-06.

Purpose . The aim of the study was, through a single institutional analysis of a large population of breast cancer patients, to assess the feasibility of and the tolerance to radiotherapy after the use of magnetic detection method for sentinel lymph node biopsy. Material and methods The super paramagnetic iron oxide particles database was collected prospectively and identified 520 cases from October 2013 to December 2016 at our institution. All of them received super paramagnetic iron oxide particles injection 20 minutes before the surgical procedure and some of them received also isotope technique. Injection site for super paramagnetic iron oxide particles and isotope was periareolar. Among them, 288 patients received adjuvant radiotherapy. In our study, we evaluated the tolerance of postoperative radiotherapy. Results The median age of the patients was 64 years. The median follow-up period was 16 months (range: 1–42 months). Double detection of sentinel lymph node was done in the first 30 patients (10.4%). The sentinel lymph node identification rate was 99.7% (287 out of 288). There were 34 axillary lymph node dissections, of which 58.8% were realized straightaway. The total radiation dose was 50 Gy EQD2 (range: 28.5–66 Gy). Regarding the occurrence of radiodermatitis, 95.8% of patients had grade 0–2 radiodermatitis and 1% had grade 3. During follow-up, 19.4% of patients developed grade 1–2 post-therapeutic fibrosis (of which 92.9% grade 1). Conclusion The results of this large-scale study show that the radiotherapy after sentinel lymph node biopsy using super paramagnetic iron oxide particles is feasible, and that no increase of the toxicity was observed.

Precision oncology is currently based on pairing molecularly targeted agents (MTA) to predefined single driver genes or biomarkers. Each tumor harbors a combination of a large number of potential genetic alterations of multiple driver genes in a complex system that limits the potential of this approach. We have developed an artificial intelligence (AI)-assisted computational method, the digital drug-assignment (DDA) system, to prioritize potential MTAs for each cancer patient based on the complex individual molecular profile of their tumor. We analyzed the clinical benefit of the DDA system on the molecular and clinical outcome data of patients treated in the SHIVA01 precision oncology clinical trial with MTAs matched to individual genetic alterations or biomarkers of their tumor. We found that the DDA score assigned to MTAs was significantly higher in patients experiencing disease control than in patients with progressive disease (1523 versus 580, P = 0.037). The median PFS was also significantly longer in patients receiving MTAs with high (1000+ P = 0.044). Our results indicate that AI-based systems, like DDA, are promising new tools for oncologists to improve the clinical benefit of precision oncology.

The development of triple-negative breast cancer (TNBC) is critically regulated by certain tumor-microenvironment-associated cells called mesenchymal stem/stromal cells (MSCs), which we and others have shown promote TNBC progression by activating pro-malignant signaling in neighboring cancer cells. Characterization of these cascades would better our understanding of TNBC biology and bring about therapeutics that eliminate the morbidity and mortality associated with advanced disease. Here, we focused on the emerging class of RNAs called long non-coding RNAs or lncRNAs and utilized a MSC-supported TNBC progression model to identify specific family members of functional relevance to TNBC pathogenesis. Indeed, although some have been described to play functional roles in TNBC, activities of lncRNAs as mediators of tumor-microenvironment-driven TNBC development remain to be fully explored. We report that MSCs stimulate robust expression of LINC01119 in TNBC cells, which in turn induces suppressor of cytokine signaling 5 (SOCS5), leading to accelerated cancer cell growth and tumorigenesis. We show that LINC01119 and SOCS5 exhibit tight correlation across multiple breast cancer gene sets and that they are highly enriched in TNBC patient cohorts. Importantly, we present evidence that the LINC01119-SOCS5 axis represents a powerful prognostic indicator of adverse outcomes in TNBC patients, and demonstrate that its repression severely impairs cancer cell growth. Altogether, our findings identify LINC01119 as a major driver of TNBC development and delineate critical non-coding RNA theranostics of potential translational utility in the management of advanced TNBC, a class of tumors in most need of effective and targeted therapy.

Expression of hormone receptor (HR) for estrogens (ER) and progesterone (PR) and HER2 remains the cornerstone to define the therapeutic strategy for breast cancer patients. We aimed to compare phenotypic profiles between matched primary and metastatic breast cancer (MBC) in the ESME database, a National real-life multicenter cohort of MBC patients. Patients with results available on both primary tumour and metastatic disease within 6 months of MBC diagnosis and before any tumour progression were eligible for the main analysis. Among the 16,703 patients included in the database, 1677 (10.0%) had available biopsy results at MBC diagnosis and on matched primary tumour. The change rate of either HR or HER2 was 27.0%. Global HR status changed (from positive = either ER or PR positive, to negative = both negative; and reverse) in 14.2% of the cases (expression loss in 72.5% and gain in 27.5%). HER2 status changed in 7.8% (amplification loss in 45.2%). The discordance rate appeared similar across different biopsy sites. Metastasis to bone, HER2+ and RH+/HER2- subtypes and previous adjuvant endocrine therapy, but not relapse interval were associated with an HR discordance in multivariable analysis. Loss of HR status was significantly associated with a risk of death (HR adjusted = 1.51, p = 0.002) while gain of HR and HER2 discordance was not. In conclusion, discordance of HR and HER2 expression between primary and metastatic breast cancer cannot be neglected. In addition, HR loss is associated with worse survival. Sampling metastatic sites is essential for treatment adjustment.

International audience; Bone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing.

Simple Summary Immune checkpoint inhibitors (ICI) are now part of the therapeutical arsenal for cancers at several sites and in several settings. PD-L1 expression is assessed to predict treatment response. We used immunohistochemistry (E1L3N clone) to assess PD-L1 expression on tumor and immune cells from a cohort of 89 surgical specimens of T1-T3NxM0 triple-negative breast cancers (TNBC) from patients treated with neoadjuvant chemotherapy (NAC) with residual disease. PD-L1 expression levels were low in tumor and immune cells from post-NAC surgical specimens. PD-L1 positivity in tumor cells was significantly associated with aggressive post-NAC tumor characteristics. A small subset of TNBC patients displaying PD-L1 expression in the context of a more extensive post-NAC tumor burden could benefit from ICI treatment after NAC. Abstract The consequences of neoadjuvant chemotherapy (NAC) for PD-L1 activity in triple-negative breast cancers (TNBC) are not well-understood. This is an important issue as PD-LI might act as a biomarker for immune checkpoint inhibitors’ (ICI) efficacy, at a time where ICI are undergoing rapid development and could be beneficial in patients who do not achieve a pathological complete response. We used immunohistochemistry to assess PD-L1 expression in surgical specimens (E1L3N clone, cutoff for positivity: ≥1%) on both tumor (PD-L1-TC) and immune cells (PD-L1-IC) from a cohort of T1-T3NxM0 TNBCs treated with NAC. PD-L1-TC was detected in 17 cases (19.1%) and PD-L1-IC in 14 cases (15.7%). None of the baseline characteristics of the tumor or the patient were associated with PD-L1 positivity, except for pre-NAC stromal TIL levels, which were higher in post-NAC PD-L1-TC-positive than in negative tumors. PD-L1-TC were significantly associated with a higher residual cancer burden (p = 0.035) and aggressive post-NAC tumor characteristics, whereas PD-L1-IC were not. PD-L1 expression was not associated with relapse-free survival (RFS) (PD-L1-TC, p = 0.25, and PD-L1-IC, p = 0.95) or overall survival (OS) (PD-L1-TC, p = 0.48, and PD-L1-IC, p = 0.58), but high Ki67 levels after NAC were strongly associated with a poor prognosis (RFS, p = 0.0014, and OS, p = 0.001). A small subset of TNBC patients displaying PD-L1 expression in the context of an extensive post-NAC tumor burden could benefit from ICI treatment after standard NAC.

VGLL2-rearranged rhabdomyosarcomas (RMS) are rare low-grade tumors with only favorable outcomes reported to date. We describe 4 patients with VGLL2-rearranged RMS confirmed by molecular studies, who experienced local progression and distant metastases, including 2 with fatal outcomes. Tumors were diagnosed at birth (n=3) or at 12 months of age (n=1), and were all localized at initial diagnosis, but unresectable and therefore managed with chemotherapy and surveillance. Metastatic progression occurred from 1 to 8 years from diagnosis (median, 3.5 y). Three patients experienced multimetastatic spread and one showed an isolated adrenal metastasis. At initial diagnosis, 3 tumors displaying bland morphology were misdiagnosed as fibromatosis or infantile fibrosarcoma and initially managed as such, while 1 was a high-grade sarcoma. At relapse, 3 tumors showed high-grade morphology, while 1 retained a low-grade phenotype. Low-grade primary tumors showed only very focal positivity for desmin, myogenin, and/or MyoD1, while high-grade tumors were heterogenously or diffusely positive. Whole-exome sequencing, performed on primary and relapse samples for 3 patients, showed increased genomic instability and additional genomic alterations (eg, TP53, CDKN2A/B, FGFR4) at relapse, but no recurrent events. RNA sequencing confirmed that high-grade tumors retained VGLL2 fusion transcripts and transcriptomic profiles consistent with VGLL2-rearranged RMS. High-grade samples showed a high expression of genes encoding cell cycle proteins, desmin, and some developmental factors. These 4 cases with distinct medical history imply the importance of complete surgical resection, and suggest that RMS-type chemotherapy should be considered in unresectable cases, given the risk of high-grade transformation. They also emphasize the importance of correct initial diagnosis.

International audience; Cancers of unknown primary (CUP) are metastatic cancers for which the primary tumor is not found despite thorough diagnostic investigations. Multiple molecular assays have been proposed to identify the tissue of origin (TOO) and inform clinical care; however, none has been able to combine accuracy, interpretability, and easy access for routine use. We developed a classifier tool based on the training of a variational autoencoder to predict tissue of origin based on RNA-sequencing data. We used as training data 20,918 samples corresponding to 94 different categories, including 39 cancer types and 55 normal tissues. The TransCUPtomics classifier was applied to a retrospective cohort of 37 CUP patients and 11 prospective patients. TransCUPtomics exhibited an overall accuracy of 96% on reference data for TOO prediction. The TOO could be identified in 38 (79%) of 48 CUP patients. Eight of 11 prospective CUP patients (73%) could receive first-line therapy guided by TransCUPtomics prediction, with responses observed in most patients. The variational autoencoder added further utility by enabling prediction interpretability, and diagnostic predictions could be matched to detection of gene fusions and expressed variants. TransCUPtomics confidently predicted TOO for CUP and enabled tailored treatments leading to significant clinical responses. The interpretability of our approach is a powerful addition to improve the management of CUP patients. Copyright

Recommendations regarding the administration of adjuvant systemic treatment to patients with invasive breast carcinoma (BC) rely on the morphological assessment of key histopathological indices including tumour subtype, size, histological grade, lymphovascular invasion and lymph node status combined with ER, PR and HER2 receptor biomarker profile determined using immunohistochemistry and ISH. The presence of tumour infiltrating lymphocytes (TILs) is becoming increasingly important in triple negative BC (TNBC). In recent years, gene expression signatures or profile (GEP) assays have been developed to help to predict the risk of recurrence and to estimate the potential benefit from adjuvant systemic chemotherapy (CT) in patients with luminal BC.

The consequences of neoadjuvant chemotherapy (NAC) for PD-L1 activity in triple-negative breast cancers (TNBC) are not well understood. This is an important issue as immune checkpoint inhibitors (ICI) are undergoing rapid development and could be beneficial in patients who do not achieve a pathological complete response. We used immunohistochemistry to assess PD-L1 expression (E1L3N clone, cutoff for positivity: ≥ 1%) on both tumor (PD-L1-TC) and immune cells (PD-L1-IC) from a cohort of surgical specimens of T1-T3NxM0 TNBCs treated with NAC. PD-L1-TC was detected in 17 cases (19.1%) and PD-L1-IC in 14 cases (15.7%). None of the baseline characteristics of the tumor or the patient were associated with PD-L1 positivity, except for pre-NAC stromal TIL levels, which were higher in post-NAC PD-L1-TC-positive than in negative tumors. PD-L1-TC were significantly associated with a higher residual cancer burden (p=0.035) and aggressive post-NAC tumor characteristics, whereas PD-L1-IC were not. PD-L1 expression was not associated with DFS (p=0.38) or OS (p=0.48), but high Ki67 levels after NAC were strongly associated with a poor prognosis (DFS p=0.0014 and OS p=0.001). A small subset of TNBC patients displaying PD-L1 expression in the context of an extensive post-NAC tumor burden could benefit from ICI treatment after standard NAC.

Purpose Precision medicine trials constitute a precious source of molecular data with prospective clinical annotations allowing the exploration of patients’ subpopulations according to specific clinical or biological questions. Using the SHIVA01—the first randomized trial comparing molecularly targeted therapy on the basis of tumor molecular profiling versus conventional chemotherapy in metastatic cancer patients who failed standard of care therapy—annotated database, we report cases of patients treated in the trial with targeted therapy who experienced an objective response or prolonged disease stabilization in light of patients’ molecular alterations. Patients and Methods We selected all patients included in SHIVA01 treated with a molecularly targeted agent (MTA) who experienced an objective response or disease stabilization that lasted longer than 6 months according to Response Evaluation Criteria in Solid Tumors version 1.1. Results Among the 170 patients who received MTAs in the SHIVA01 trial, 15 patients (9%) experienced an objective response (n = 3) or disease stabilization that lasted longer than 6 months (n = 12). The most frequent histologic subtypes were breast cancer (27%) and cervical cancer (20%). Six patients, including three patients with breast cancer, were treated with abiraterone on the basis of androgen receptor protein overexpression. Five patients were treated with everolimus on the basis of a PTEN heterozygous deletion with loss of protein expression, PIK3CA mutation, or both alterations. The remaining four patients were treated with tamoxifen, erlotinib, imatinib, and vemurafenib on the basis of progesterone receptor expression, EGFR amplification, KIT mutation, and BRAF mutation, respectively. TP53 mutations were absent in responder patients. Conclusion Analysis of patients who experienced objective responses or disease stabilization that lasted longer than 6 months allowed the identification of potential biomarkers of sensitivity and resistance to MTAs.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment., Identifying novel therapies for the treatment of CDK4/6 inhibitor-resistant patients is of great importance. Here, the authors demonstrate that PLK1 inhibition is a potential therapeutic target in CCND1-driven and in RB-positive Palbociclib-resistant breast cancers.

Background High levels of stromal tumor-infiltrating lymphocytes (sTIL) are associated with increased pathological complete response (pCR) rate and longer survival after neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) patients. Here, we evaluated the value of sTIL in predicting pCR and explored prognosis in TNBC patients treated with neoadjuvant chemotherapy according to body mass index (BMI). Methods sTIL were scored centrally on pretreatment biopsies from 2 retrospective series of nonunderweight TNBC patients (n = 445). sTIL and BMI were considered as binary (sTIL: Results 236 (53.0%) patients were lean and 209 (47.0%) overweight and obese. pCR was achieved in 181 of 445 (41.7%) patients. Median sTIL was 11.0%, and 99 of 445 (22.2%) tumors had high sTIL. A statistically significant interaction between sTIL and BMI, considered as categorical or continuous variables, for predicting pCR was observed in the multivariable analysis (Pinteraction = .03 and .04, respectively). High sTIL were statistically significantly associated with pCR in lean (odds ratio [OR] = 4.24, 95% confidence interval [CI] = 2.10 to 8.56; P < .001) but not in heavier patients (OR = 1.48, 95% CI = 0.75 to 2.91; P = .26) in the multivariable analysis. High sTIL were further associated with increased event-free survival in lean (hazard ratio [HR] = 0.22, 95% CI = 0.08 to 0.62; P = .004) but not in heavier patients (HR = 0.53, 95% CI = 0.26 to 1.08; P = .08). Similar results were obtained for overall survival. Conclusion BMI is modifying the effect of sTIL on pCR and prognosis in TNBC patients treated with neoadjuvant chemotherapy.

Tumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy., Tumor-draining lymph nodes are often the first site of metastasis in breast cancer patients. Here, the authors show that metastatic lymph nodes are characterized by the accumulation of suppressive regulatory T cells with a distinct phenotype compared to matched non-invaded lymph nodes and tumors.

Invasive lobular carcinoma (ILC) represents the second most common histologic breast cancer subtype after invasive ductal carcinoma (IDC). While primary ILC has been extensively studied, metastatic ILC has been poorly characterized at the genomic and immune level., info:eu-repo/semantics/published

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls., info:eu-repo/semantics/published