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Background: All intrinsic molecular subtypes are represented among HER2-positive breast cancer, with implications on clinical outcome and treatment sensitivity. The impact of molecular subtypes on the pattern and site of relapse is largely unexplored. Methods: 677 patients with HER2-positive early breast cancer from the Shorther trial (n=437), the CherLOB trial (n=84) and two Institutional cohorts (Istituto Oncologico Veneto IRCCS Padova n=39 and Hospital Clinic Barcelona n=117) were included. Only patients with available PAM50 intrinsic molecular subtyping were considered. We analyzed the incidence of distant relapse (at any site and at specific sites) as the first event. Cumulative incidence was estimated according to competing risk analysis (Fine and Gray’s method). Competing risk regression was used to calculate the subdistribution Hazard Ratios (subHR) and their 95% Confidence Interval (CI). Results: The distribution of molecular subtypes was: 130 LumA (19%), 75 LumB (11%), 347 HER2-e (51%), 46 Basal (7%), 79 Normal (12%). Median follow up was 8.4 years (95%CI 8.2-8.6). The 10-yr cumulative incidence rates of distant relapse as first event were: LumA 7.9%, LumB 14.8%, HER2-e 14.7%, Basal 15.5%, Normal 10.4% (HER2-e vs LumA: SubHR 2.21, 95%CI 1.05-4.64, p=0.037). Table 1 shows the 5-yr and 10-yr cumulative incidence rates of distant metastases at specific sites (as first event) according to intrinsic subtype. HER2-e enriched and Basal tumors were more prone as compared to other subtypes to develop brain and lung metastasis as first event, respectively. Isolated brain metastases without extracranial disease occurred only in patients with HER2-e tumors. All brain metastases as first event occurred within 5 years from diagnosis. Bone-only disease as first event was less frequent in HER2-e and Basal subtype (subHR HER2-e vs LumA: 0.32, 95%CI 0.10-10.4. p=0.058). Next, we analyzed the frequency of site-specific first metastasis among patients who experienced a distant metastasis as first event (n=77). Lung metastases were more frequent in Basal tumors (LumA 25.0%, LumB 20.0%, HER2-e 24.4%, Basal 71.4%, Normal 0.0%, p=0.037) and bone metastases were more frequent in Luminal tumors (LumA 100.0%, LumB 60.0%, HER2-e 31.1%, Basal 42.9%, Normal 57.1%, p=0.006). Among 45 HER2-e patients with a first distant relapse, 25.6% were diagnosed with a brain metastasis and 15.6% had brain-only disease. Conclusions: Molecular subtypes influence the metastatic behaviour of clinically HER2-positive breast cancer. These results, if further validated, may have implication in planning personalized monitoring strategies. Table 1. 5-yr and 10-yr cumulative incidence rates of distant metastasis at specific sites (as first event) according to intrinsic subtype. Citation Format: Maria Vittoria Dieci, Giancarlo Bisagni, Stefania Bartolini, Antonio Frassoldati, Daniele Giulio Generali, Federico Piacentini, Gaia Griguolo, Enrico Tagliafico, Fara Brasó-Maristany, Nuria Chic, Francesca Porra, Roberto Vicini, Roberto D’Amico, Sara Balduzzi, Aleix Prat, PierFranco Conte, Valentina Guarneri. Pattern of distant relapse according to intrinsic molecular subtype in patients with HER2-positive breast cancer: a combined analysis of ShortHER, CherLOB, and two institutional cohorts. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-12.

Background: Extending adjuvant endocrine treatment (ET) with aromatase inhibitors (AI) to 7-10 years decreases the risk of relapse in hormone receptor-positive (HR+) breast cancer (BC). However, such benefit comes at the price of higher incidence of skeletal and cardiovascular (CV) events. Biomarkers predicting such toxicities might help clinicians in tailoring adjuvant ET to patient’s needs. We conducted a prospective study to assess whether SNPs in the gene encoding for the aromatase enzyme (CYP19A1) affect the risk of skeletal and CV events in HR+ early BC patients enrolled in the GIM4 trial. Methods: The GIM4 trial randomized HR+ BC postmenopausal patients who had been already treated with 2-3 years of adjuvant tamoxifen to either 3-2 years or 5 years of adjuvant letrozole. Four SNPs of CYP19A1 were evaluated: rs10046, rs4646, rs479292 and rs727479. SNPs were genotyped through PCR on DNA obtained from patients’ peripheral blood samples. Skeletal and CV events were assessed from randomization in the GIM4 trial to last follow-up, disease recurrence or death. Skeletal events were defined as the onset of osteoporosis or bone fractures. CV events were defined as the onset of thrombosis, embolism, stroke, myocardial infarction, hearth failure, or arrythmia. Univariate and multivariate logistic regressions were performed to evaluate the association between SNPs and skeletal and CV events. Bonferroni correction for multiplicity was used for univariate SNPs association tests, with a corrected alpha of 0.012. Only associations with an alpha level Citation Format: Benedetta Conte, Chiara Molinelli, Giancarlo Bisagni, Antonio Durando, Giovanni Sanna, Stefania Gori, Ornella Garrone, Stefano Tamberi, Sabino De Placido, Francesco Schettini, Antonio Pazzola, Riccardo Ponzone, Filippo Montemurro, Gianluigi Lunardi, Rosario Notaro, Anna Turletti, Claudia Bighin, Francesca Poggio, Giulia Buzzatti, Matteo Lambertini, Luca Boni, Lucia Del Mastro. Single nucletotide polymorphisms of aromatase gene (CYP19A1) and toxicity of adjuvant aromatase inhibitors: A translational, prospective study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-03.

IntroductionPatients with triple-negative breast cancer (TNBC) achieving a pathological complete response (pCR) after neoadjuvant chemotherapy have a better event-free survival. The role of gut microbiome in early TNBC is underexplored.MethodsMicrobiome was analyzed by 16SrRNA sequencing.ResultsTwenty-five patients with TNBC treated with neoadjuvant anthracycline/taxane-based chemotherapy were included. Fifty-six percent achieved a pCR. Fecal samples were collected before (t0), at 1 (t1), and 8 weeks (t2) from chemotherapy. Overall, 68/75 samples (90.7%) were suitable for microbiome analysis. At t0, pCR group showed a significantly higher α-diversity as compared with no-pCR, (P = .049). The PERMANOVA test on β-diversity highlighted a significant difference in terms of BMI (P = 0.039). Among patients with available matched samples at t0 and t1, no significant variation in microbiome composition was reported over time.ConclusionsFecal microbiome analysis in early TNBC is feasible and deserves further investigation in order to unravel its complex correlation with immunity and cancer.

Purpose: The role of immunotherapy in hormone receptor (HR)-positive, HER2-negative breast cancer is underexplored. Patients and Methods: The neoadjuvant phase II GIADA trial (NCT04659551, EUDRACT 2016-004665-10) enrolled stage II–IIIA premenopausal patients with Luminal B (LumB)-like breast cancer (HR-positive/HER2-negative, Ki67 ≥ 20%, and/or histologic grade 3). Patients received: three 21-day cycles of epirubicin/cyclophosphamide followed by eight 14-day cycles of nivolumab, triptorelin started concomitantly to chemotherapy, and exemestane started concomitantly to nivolumab. Primary endpoint was pathologic complete response (pCR; ypT0/is, ypN0). Results: A pCR was achieved by 7/43 patients [16.3%; 95% confidence interval (CI), 7.4–34.9]; the rate of residual cancer burden class 0–I was 25.6%. pCR rate was significantly higher for patients with PAM50 Basal breast cancer (4/8, 50%) as compared with other subtypes (LumA 9.1%; LumB 8.3%; P = 0.017). Tumor-infiltrating lymphocytes (TIL), immune-related gene-expression signatures, and specific immune cell subpopulations by multiplex immunofluorescence were significantly associated with pCR. A combined score of Basal subtype and TILs had an AUC of 0.95 (95% CI, 0.89–1.00) for pCR prediction. According to multiplex immunofluorescence, a switch to a more immune-activated tumor microenvironment occurred following exposure to anthracyclines. Most common grade ≥3 treatment-related adverse events (AE) during nivolumab were γ-glutamyltransferase (16.7%), alanine aminotransferase (16.7%), and aspartate aminotransferase (9.5%) increase. Most common immune-related AEs were endocrinopathies (all grades 1–2; including adrenal insufficiency, n = 1). Conclusions: Luminal B-like breast cancers with a Basal molecular subtype and/or a state of immune activation may respond to sequential anthracyclines and anti–PD-1. Our data generate hypotheses that, if validated, could guide immunotherapy development in this context.

Background: Hormone receptor positive (HR+)/HER2- breast cancer (BC) is a clinically and biologically heterogeneous disease, encompassing all BC molecular intrinsic subtypes. We here explore the association between gene-expression profiles and response to neoadjuvant endocrine-based treatment in early HR+/HER2- BC. Methods: The LETLOB phase II trial randomized 92 postmenopausal women with clinical stage II-IIIA HR+/HER2- BC to receive neoadjuvant letrozole + lapatinib or letrozole + placebo for 6 months. Objective response was the primary endpoint, as defined by study protocol and previously published (Guarneri, JCO 2014). Gene-expression data (Affymetrix platform) from pre-treatment frozen core-biopsies was available for 66 pts. Intrinsic subtype was assigned using a research-based PAM50 subtype predictor. The PAM50 based chemo-endocrine score (CES) was calculated using published definition (Prat, CCR 2017). Higher values of CES indicate increased endocrine sensitivity, while lower values indicate higher chemosensitivity. Results: Intrinsic subtype distribution was as follows: Luminal A 39% (N=25), Luminal B 36% (N=24), HER2-enriched 8% (N=5), basal-like 18% (N=12). Non-luminal tumors (basal-like and HER2-enriched) were characterized by less differentiated histological grades (Grade 3 65% vs 33%, p=0.037) and higher Ki67 expression at baseline as compared to luminal tumors (median 20% vs 15%, p=0.013). Considering both treatment arms combined, non-luminal tumors showed a significantly lower objective response rate as compared to luminal tumors (47% vs 78%, p=0.031). This difference was statistically significant in the lapatinib arm (43% vs 90%, p=0.021), but not in the placebo arm (50% vs 68%, p=0.449; interaction test p=0.169). After treatment, non-luminal tumors had significantly higher Ki67 levels than luminal ones (post-treatment median 10% and 7%, respectively, p=0.004). Median CES level at baseline was -0.03 (range: -1.11, +1.12). Each unit increase in CES levels at baseline was numerically associated with higher probability of achieving an objective response (Odds Ratio 1.95, 95% CI 0.74-5.18, p=0.178, both arms combined). Conclusions: In HR+/HER2- early BC patients enrolled in the LET-LOB trial, patients with non-luminal tumor or low CES score showed reduced sensitivity to endocrine-based treatment. The highest response rate (90%) was observed in luminal patients treated with letrozole + lapatinib. Gene-expression profiling could potentially be used to identify patients with low sensitivity to endocrine treatment for which neoadjuvant chemotherapy should be preferred. Citation Format: Gaia Griguolo, Maria Vittoria Dieci, Daniele Giulio Generali, Laia Paré, Antonio Frassoldati, Luigi Cavanna, Enrico Tagliafico, Katia Cagossi, Giancarlo Bisagni, Federico Piacentini, Federica Miglietta, Guido Ficarra, Aleix Prat, PierFranco Conte, Valentina Guarneri. Gene-expression profiling and response to neoadjuvant endocrine treatment in the phase II LETLOB trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-14.

Background Despite endocrine therapy is the mainstay of treatment for HR+/HER2- metastatic breast cancer (MBC), patients with high disease burden or those at risk of visceral crisis are still offered first-line chemotherapy (CT). Chemotherapy is generally followed by maintenance hormonal therapy. The MAIN-A study is an investigator-driven, randomized phase III trial designed to compare maintenance everolimus (EVE) combined with aromatase inhibitors (AI) versus AI alone in pts with disease control after first-line CT.Methods Postmenopausal pts achieving disease control (stable disease, partial response or complete response) after first-line CT were randomly assigned to EVE 10 mg po daily plus AI or to AI alone. Primary aim was PFS in the ITT. We present here overall survival (OS) results and the impact of tumor characteristics on PFS.Results 110 pts were randomized to EVE+AI (n=52) or to AI (n= 58). Primary aim results have been already presented (Guarneri V, ESMO Breast 2019), showing a non-significant prolongation of median PFS in the ITT population for EVE+AI (9.9 mos vs 7.2 mos, HR 0.764, 95% CI 0.501-1.164. Patients with visceral metastases tended to experience shorter PFS as compared with patients with patients with bone/soft tissues metastases (median 11.1 mos vs 6.4 mos, p=0.0746). The levels of estrogen receptor expression (>or< 50%) did not impact PFS, overall and by treatment arm. At the time of this writing, a total of 61 death events have been recorded. No difference in OS was observed between the two arms (median 33.9 mos for EVE+AI vs 33.5 mos for AI, HR 0.97, 95% CI 0.59-1.61).Conclusions Maintenance EVE+AI did not significantly impact on the outcome of metastatic breast cancer patients deemed suitable for first line chemotherapy. Citation Format: Valentina Guarneri, Saverio Cinieri, Maria Vittoria Dieci, Carmelo Bengala, Gabriella Mariani, Giancarlo Bisagni, Antonio Frassoldati, Claudio Zamagni, Laura Orlando, Carlo Alberto Giorgi, Gian Luca De Salvo, PierFranco Conte. Maintenance therapy with everolimus plus aromatase inhibitors vs aromatase inhibitors as after first-line chemotherapy in HR+/HER2- metastatic breast cancer: Updated analyses of the phase III randomized MAIN-A trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-22.

Background: Tumor-infiltrating lymphocytes (TILs) have been reported to be associated with increased therapeutic efficacy of trastuzumab/pertuzumab-based neoadjuvant therapy (NT) in patients (pts) with HER2-positive breast cancer (BC). Subcutaneous (SC) trastuzumab has non-inferior efficacy to intravenous (IV) administration, with a similar safety profile. Interestingly, SC trastuzumab has been observed to be more immunogenic than IV trastuzumab and act at different immunologic levels. Therefore, by modifying the modality of administration of trastuzumab, it could be possible to interfere with different immune pathways and exert a favorable immunomodulation in HER2-positive BC. Methods: In this non-comparative, phase II, neoadjuvant, randomized study, pts were eligible if they had previously untreated, histologically confirmed, locally advanced, inflammatory, or early-stage HER2-positive BC. Pts were treated with FEC (fluorouracil 500 mg/m2; epirubicin 75 mg/m2; cyclophosphamide 500 mg/m2) q21 × 3 cycles. Then, they were randomly assigned (1:1) to receive: docetaxel (75 mg/m2) plus pertuzumab (840 mg loading dose (LD), then 420 mg) plus IV trastuzumab (8 mg/kg LD, then 6 mg/kg) q21 × 4 cycles (arm A) or, docetaxel plus pertuzumab plus SC trastuzumab (fixed dose of 600 mg) q21 × 4 cycles (arm B). After surgery, pts received trastuzumab q21 × 14 cycles using the same formulation (SC or IV) of the preoperative phase. The primary endpoint was the rate of stromal TILs (sTILs) on residual disease after surgery. Tumor biopsy and posttreatment surgical samples were centrally analyzed for TILs. Blood samples were also collected during NT for tumor-specific lymphocyte cell activity analysis. Feasibility, efficacy and safety were also evaluated. ClinicalTrials.gov: NCT03144947. Results: Between November 2016 and September 2017, according to an adaptive Simon's two-stage optimal design, we enrolled 65 pts, of whom two were deemed ineligible for the study. Thus, 63 pts (31 in arm A and 32 in arm B) were assessed for the primary and secondary endpoints. The pathologic complete response (pCR; no invasive tumor in breast and axilla) rates were 64.5% (95% CI, 47-81) in arm A, and 59.4% (95% CI, 42-76) in arm B. The most common adverse events of grade 3 or higher were neutropenia (15 [48.4%] pts in arm A, and 11 [34.4%] in arm B), neurotoxicity (1 [3.2%], and 2 [6.2%], respectively), and diarrhea (1 [3.2%], and 1 [3.1%], respectively). There were no events of congestive heart failure. At surgery, 11 pts in arm A and 13 pts in arm B were evaluable for TIL analysis. The median value of sTILs (7.5%) on pre-treatment tumor biopsies was used as the cut-off value, and high sTIL levels were observed in 27.3% and in 46.1% of residual tumors after treatment arm A and B, respectively. There was a positive correlation between pretreatment sTILs and PD-L1 expression on stromal immune cells (Kendall’s τ =0.80). Interestingly, a significant inverse correlation was observed between PD-L1 expression on pretreatment sTILs and the T cell co-receptor CD3 expressed on posttreatment sTILs (Pearson’s ρ = -0.70). This finding was particularly evident in the arm B group (ρ = -0.85). Conclusions: NT with either SC or IV trastuzumab in combination with pertuzumab and chemotherapy had a significant effect on sTIL expression at surgery. In particular, the SC trastuzumab-based arm exerted the most relevant enrichment of sTILS in posttreatment residual tumors. These findings suggest a role for the SC administration of trastuzumab in determining favorable variations of host immune response parameters among pts with HER2-positive early BC who had residual disease after NT. Citation Format: Antonino Musolino, Stefania Gori, Elisabetta Cretella, Alessandra Marabese, Luigi Cavanna, Antonio Frassoldati, Giancarlo Bisagni, Chiara Casarini, Emilio Bria, Luisa Carbognin, Elena Fiorio, Alba A Brandes, Claudio Zamagni, Lorenzo Gianni, Alberto Zambelli, Filippo Montemurro, Michele Tognetto, Renata Todeschini, Giuseppe Maglietta, Gabriele Missale, Enrico M Silini. Phase II, open label, randomized, biomarker study of immune-mediated mechanism of action of neoadjuvant subcutaneous trastuzumab in patients with operable, locally advanced, or inflammatory HER2-positive breast cancer. ImmunHER trial on behalf of the Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-19.

Background We assessed TILs before and during treatment in patients with ER+/HER2+ breast cancer (BC) enrolled in the NA-PHER2 study, and evaluated the association between TILs and their modulation over treatment with likelihood of achieving a pCR. Methods In the NA-PHER2 study (NCT02530424), patients with centrally confirmed ER+ (>10%) HER2+ BC were treated in two independent, non-randomized, cohorts with neoadjuvant trastuzumab, pertuzumab, palbocilib with (Cohort A, n=30) or without (Cohort B, n=28) fulvestrant (+/-LHRH analogues). We measured TILs by optical evaluation on core-biopsies obtained pre-treatment [pre-CBX, n=56/58 (96.5%)], at 14 days if invasive tumor cells [14d-CBX, n=52/53 (98.1%)], and on residual disease at surgery [SX, n=45/45 (100%)]. We scored TILs as stromal and intratumoral TILs (sTILs, iTILs), and hotspot TILs (hTILs) i.e. the fields of highest TILs density. We investigated the association between baseline and 14 days TILs with pCR, including the early TILs changes (pre vs 14 days) [delta TILs (ΔTILs) was the absolute percentage change). TILs biomarkers were assessed as continuous variable (10% increase). Results The three TILs metrics were significantly correlated at all time-points (all p0.55). TILs metrics were not associated with T and N stage, PgR status. sTILs were correlated with Ki67 as continuous variable at 14 days (corr 0.41 p=0.003) and surgery (corr 0.34, p=0.025), but not at baseline. At baseline (pre-CBX), only high sTILs were associated with pCR (OR 1.55 [1.01-2.37], p=0.044). At 14 days (on treatment), all TILs assessments were associated with pCR (OR 1.96 [1.16-3.31], p=0.011 for sTILs; OR 5.85 [1.39-24.6], p=0.016 for iTILs; OR 1.58 [1.16-2.14], p=0.004). When combined in multivariate model, they did not provide independent information. Considering early changes of TILs (baseline vs 14 days), only ΔiTILs was associated with pCR (OR 3.09 [1.50-6.38], p=0.002). When baseline, 14 days and dynamic biomarker was assessed in multivariate, only ΔiTILs retained the significance. The predictive value of TILs was similar in the two treatment cohorts. Overall, there was not average difference between any TILs assessment method comparing the three time points. By evaluating only patients with RD and TILs assessment available at all three time points (n=41/45, 91.1%), some intra-patient variability emerged. ΔTIL higher than 10% was observed in 13/41 (31.7%) cases. Seven (17.1%) had an increase, 4 (9.7%) a mixed behavior and 2 (4.9%) had a decrease. Dynamic change of TILs was not associated with change in Ki67. When TILs infiltration in the two treatment cohort was compared at different time points, it was different only at surgery, when sTILs and iTILs were lower in patients treated with Fulvestrant in Cohort A (p=0.19 and p=0.013, respectively). Conclusions In the NA-PHER2 study, we show that in ER+/HER2+ BC the likelihood of achieving pCR using a chemo-free regimen is larger in patients with higher sTILs infiltration. TILs assessment on core biopsies after 14 days was also predictive of pCR, and early changes of iTILs was the most informative predictor suggesting that treatment may facilitate T cell infiltration in agreement with the postulated contribution of CDK4/6 to immune evasion (Goel S Nature 2017, Jerby-Arnon L Cell 2018). No average modulation of immune biomarkers was found. However, individual pts modulation suggests that immune effect might be relevant also for some patients not achieving a pCR. Notably, the presence of endocrine treatment may contribute to dampen immune activation. Changes of Ki67 and TILs were not associated suggesting that they capture different biological phenomena. Supported in part by an unrestricted grant from Pfizer Italia S.r.l. and from Roche S.p.a. Italia Citation Format: Giampaolo Bianchini, Chanel Smart, Giancarlo Bisagni, Marco Colleoni, Mauro Mansutti, Claudio Zamagni, Lucia Del Mastro, Stefania Zambelli, Luca Licata, Antonio Frassoldati, Giuseppe Viale, Pinuccia Valagussa, Luca Gianni. Modulation of tumor infiltrating lymphocytes (TILs) by palbociclib, trastuzumab, pertuzumab +/- fulvestrant in ER+/HER2+ patients (pts) enrolled in the Michelangelo NA-PHER2 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-14-01.

BackgroundDerived neutrophil-to-lymphocyte ratio (dNLR) is a biomarker associated with clinical outcome in breast cancer (BC). We analyzed the association of dNLR with pathological complete response (pCR) in triple-negative BC (TNBC) patients receiving neoadjuvant chemotherapy (CT).MethodsThis is a retrospective analysis of two randomized studies involving early stage/locally advanced TNBC patients receiving anthracycline/taxane-based CT+/−carboplatin (GEICAM/2006-03) or nab-paclitaxel/paclitaxel followed by anthracycline regimen (ETNA). dNLR was calculated as the ratio of neutrophils to the difference between total leukocytes and neutrophils in peripheral blood before CT (baseline) and at the end of treatment (EOT). Logistic regression analyses were used to explore dNLR association with pCR.ResultsIn total, 308 TNBC patients were analyzed, 216 from ETNA and 92 from GEICAM/2006-03. Baseline median dNLR was 1.61 (interquartile range (IQR): 1.25–2.04) and at EOT 1.53 (IQR: 0.96–2.22). Baseline dNLR showed positive correlation with increased tumor size (p-value = 1e−04). High baseline dNLR, as continuous variable or using median cutoff, was associated with lower likelihood of pCR in univariate analysis. High EOT dNLR as continuous variable or using quartiles was also associated with lower pCR rate in uni- and multivariate analyses.ConclusionsHigh baseline and EOT dNLR correlates with lower benefit from neoadjuvant CT in TNBC.

Background: Hormone-receptor (HR)+/HER2- breast cancer (BC) is a biologically heterogeneous disease. Homologous recombination deficiency (HRD) and BRCA mutations have been previously reported to be associated with worse outcomes in HR+/HER2- metastatic BC patients receiving CDK4/6 inhibitors and endocrine therapy. Here, we assess the relation between HRD and RB-loss signatures, intrinsic subtyping, the PAM50-based chemo-endocrine score, and response to chemotherapy-based therapy and endocrine treatment in HR+/HER2- early BC. Methods: GIADA is a multicentric neoadjuvant phase II trial that treated premenopausal patients with Luminal B (LumB)-like BC (HR-positive, HER2-negative, with Ki67>20% and/or histologic Grade 3) with a combination of chemotherapy, immunotherapy and endocrine treatment. Expression of 758 genes on baseline tumor samples from all 43 patients was quantified by nCounter platform. The LETLOB phase II trial randomized postmenopausal women with clinical stage II-IIIA HR+/HER2- BC to neoadjuvant letrozole + lapatinib or letrozole + placebo for 6 months (Guarneri, JCO 2014). Gene-expression data (Affymetrix platform) from pre-treatment frozen core-biopsies was available from 66 out of 92 pts enrolled. Intrinsic subtype was assigned using a research-based PAM50 subtype predictor. A published HRD signature (Peng, Nat Commun 2014) and a signature of RB loss (RBsig), previously reported to potentially predict resistance to CDK4/6 inhibitors in HR+/HER2- BC (Malorni, Oncotarget 2016) were computed. The PAM50 based chemo-endocrine score (CES) was calculated using published definition (Prat, CCR 2017). Higher values of CES indicate increased endocrine sensitivity, while lower values indicate chemosensitivity. Association between genomic signatures was assessed through Pearson’s correlation coefficient. Association of genomic signatures with pCR was assessed through logistic regression and association with PEPI scores was assessed through Kruskal-Wallis test. Results: HRD signature levels were significantly higher in non-luminal (Basal-like and HER2-enriched) tumors as compared to Luminal (A or B) tumors (p>0.001 in the GIADA trial, p=0.021 in the LETLOB trial). Moreover, higher levels of HRD signature were associated with higher levels of RB-loss signature (Pearson correlation 0.355, p=0.020 in the GIADA trial; Pearson correlation 0.942, p< 0.001 in the LETLOB trial), higher levels of Basal-like signature (Pearson correlation 0.502, p< 0.001 in the GIADA trial; Pearson correlation 0.373, p=0.002 in the LETLOB trial) and lower levels of CES (Pearson correlation -0.422, p=0.005 in the GIADA trial; Pearson correlation -0.763, p< 0.001 in the LETLOB trial), indicative of higher chemosensitivity. In the GIADA trial, higher levels of HRD signature (p=0.018) and RBloss signature (p=0.073) and lower levels of CES (p=0.007) were associated with higher pCR rates after chemo, endocrine and immunotherapy. In the LETLOB trial, lower levels of HRD signature (p=0.068) and RBloss signature (p=0.042) and higher levels of CES (p=0.050) were associated with higher sensitivity to endocrine treatment (lower PEPI scores, 0 vs 1-3 vs 4 or more, after neoadjuvant letrozole). Conclusions: In HR+/HER2- early BC, HRD gene signatures, RB-loss gene signatures and non-luminal (especially Basal-like) intrinsic subtyping are associated with each other and associated with higher sensitivity to chemotherapy-based therapy and lower sensitivity to endocrine treatment. These observations might help correctly tailor systemic therapy, including biologic agents, in patients with HR+/HER2- early and advanced BC. Citation Format: Gaia Griguolo, Federica Miglietta, Laia Paré, Daniele G. Generali, Antonio Frassoldati, Antonino Musolino, Simon Spazzapan, Grazia Vernaci, Tommaso Giarratano, Marcello Lo Mele, Giancarlo Bisagni, Federico Piacentini, Enrico Tagliafico, Katia Cagossi, Francesca Schiavi, Claudia Pinato, Aleix Prat, Valentina Guarneri, Maria Vittoria Dieci. Homologous recombination deficiency, RB-loss gene signatures, intrinsic subtype and response to neoadjuvant treatment in HR+/HER2- early breast cancer: a correlative analysis of two phase II trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-13.

Introduction: More than 2 years after the WHO declaration of a pandemic, SARS-Cov-2 still represents a public health problem The pandemic has increased the complexity of cancer treatments including breast cancer. These difficulties were highlighted in adjuvant treatments but above all in metastatic disease. Vaccination has been one of the most important public health factors that has reduced deaths, hospitalizations and the severity of symptoms related to infection. In metastatic breast cancer hormone receptor positive and HER2/neu negative currently the first line of treatment is given by the association between cyclin 4/6 inhibitors and hormone therapy (aromatase inhibitors or fulvestrant) A well-known and frequent side effect of this therapy is the reduction of white blood cell values and neutrophils. The hypothesis that this study is to evaluate whether treatment with cyclin inhibitors initiated before the period of vaccinations may have influenced, due to the reduction in white blood cell values, an increased risk of infection in these patients. Materials and methods: In our study, we selected patients who had started treatment with cyclin inhibitors before starting the vaccination cycle (in Italy up to the fourth dose in cancer patients) and continue it without evidence of disease progression. All patients were offered a vaccination cycle with mRNA COVID vaccines and were followed during their cancer treatments. All patients, at least 90 days after the last dose of vaccine, have been tested for antibodies against SARS CoV-2 (trimeric spike protein) with a value expressed in binding antibodies unit (BAU) according to international standard WHO During the observation period (starting from the first dose of vaccine administered) the patients were clinically checked and in case of suspicion of infectious pathology with symptoms suggestive of SARS-COV-19 infection, they were tested with molecular swab Results: We evaluated 52 patients who started cyclin treatment before the vaccination course and who are currently without signs of disease recurrence During the study period we found 14 SARS-COV19 infections (28% of patients) and one patient with two infectious episodes. No patients needed treatment in a hospital or resuscitation setting. All patients have fully recovered from the infection and at most after 21 days have resumed the treatment still in place Statistically, a linear regression calculation was applied to evaluate a functional relationship between variables measured on the basis of sample data. We did not find a relationship between spikes or infections compared to the start date of the vaccination cycle; instead we observed a relationship between the value of the spike and the date of last immunization (considered as an active infection or fourth dose of vaccine) with a reduction in the values the further you go away Conclusion: The data of the study show that there is a correlation between the time elapsed between the last vaccination and the risk of getting sick. For this reason, the fourth recall represents a strong help to reduce this risk. We did not find any ranges we could refer to regarding the dosage of trimeric spike protein. Considering the positivity rate of infections that does not exceed the general vaccinated population and the absence of serious infectious symptoms with hospitalization, treatment with cyclin inhibitors appears to be a safe treatment even in a pandemic period. Last day immunization and spike with IA or fulvestrant Last day immunization and spike with IA or fulvestrant Citation Format: Filippo Giovanardi, Edoardo Carretto, Giancarlo Bisagni, Claudia Degli Esposti, Elisa Gasparini, Alessandra Bologna, Roberto Di Cicilia, Gabriella Moretti, Carmine Pinto. Cyclin-dependent kinase 4/6 inhibitor in advanced breast cancer during the COVID pandemic period: efficacy in relation to vaccination for SARS-COV 19 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-03-04.

This abstract was not presented at the conference. Citation Format: Gasparini E, Bisagni A, Di Cicilia R, Kuhn E, Falco G, Ferrari G, Foroni M, Tamagnini I, Bassano C, Ragazzi M, Gardini G, Cecchi F, Hembrough T, Bisagni G. Not presented [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 P3-10-24.

Background: A combined biomarker based on HER2-enriched subtype (HER2-E) and ERBB2 mRNA predicts response and survival in HER2+ breast cancer following trastuzumab +/- lapatinib in the absence of chemotherapy (Prat et al. ASCO 2018). Here, we tested the ability of the combined biomarker to predict pathological complete response (pCR) following neoadjuvant trastuzumab, pertuzumab and endocrine therapy. Methods: RNA from 40 baseline tumor samples from the phase II PerELISA trial were evaluated. PerELISA evaluated the efficacy of a de-escalated, chemotherapy-free neoadjuvant regimen based on dual HER2 blockade with trastuzumab and pertuzumab in combination with letrozole in HER2+/hormone receptor-positive breast cancer selected on the basis of Ki67 response after short course letrozole-alone (Guarneri ASCO 2018). Ki67 response was defined by protocol as relative Ki67 reduction ≥20% from baseline at day 14. Gene-expression was measured using the nCounter platform. Intrinsic subtypes and ERBB2 levels were determined by the PAM50 gene expression predictor. A pre-specified ERBB2 cutoff was determined to define ERBB2-high. Univariate and multivariable logistic regression analyses were performed. Results: The proportion of HER2-E disease within the ERBB2-high and ERBB2-low groups was 46.2% (6/13) and 18.5% (5/27), respectively. The discordance rate at the individual level was 30% (12/40). A total of 6 (15%) and 34 (85%) samples were HER2-E/ERBB2-high and others, respectively. The magnitude of Ki67 reduction of the HER2-E/ERBB2-high and others groups was 64.8% and 63.2%, respectively (p=0.88). The pCR rate of HER2-E/ERBB2-high was 66.7%. The pCR rate of the others group was 14.7%. The univariate odds ratio between HER2-E/ERBB2-high tumors and the others groups was 11.60 (95% CI 1.66-81.10; p=0.014). No other clinical-pathological variable was significantly associated with pCR. Conclusion: The combined HER2-E/ERBB2-high biomarker can identify patients who might be good candidates to receive dual HER2 blockade alone without chemotherapy. Citation Format: Prat A, Griguolo G, Dieci MV, Bisagni G, Frassoldati A, Bianchi GV, Pascual T, Pare L, Galvan P, Urso L, Conte P, Guarneri V. Independent validation of a combined biomarker based on the PAM50 HER2-enriched subtype and ERBB2 mRNA levels following HER2 blockade without chemotherapy in the PerELISA phase II 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 P6-17-05.

Aim The Cher-LOB randomised phase II study showed that the combination of lapatinib-trastuzumab plus chemotherapy increases pathologic complete response (pCR) rate compared with chemotherapy plus either trastuzumab or lapatinib. Here, we report the post hoc survival analysis as per treatment arm, pCR and biomarkers. Methods The Cher-LOB study randomised 121 patients with human epidermal growth factor receptor 2–positive, stage II–IIIA breast cancer. A specific protocol to collect recurrence-free survival (RFS) and overall survival (OS) data was designed. Tumour-infiltrating lymphocytes (TILs) and PAM50-intrinsic subtyping were evaluated at baseline. Results At 9-year median follow-up, a trend towards RFS improvement with lapatinib-trastuzumab over trastuzumab was observed (hazard ratio [HR] 0.44, 95% confidence interval [CI] 0.18–1.05). Combining treatment arms, pCR was significantly associated with both RFS (HR 0.12, 95% CI 0.03–0.49) and OS (HR 0.12, 95% CI 0.03–0.49). TILs were significantly associated with RFS (HR = 0.978 for each 1% increment). Luminal-A subtype was a significant and independent predictor of improved RFS as compared with other PAM50-based intrinsic subtypes at the multivariate analysis including the most relevant clinicopathologic variables (HR 0.29, 95% CI 0.09–0.94, p = 0.040). Conclusions Cher-LOB trial survival analysis confirmed the prognostic role of pCR and TILs and showed a signal for a better outcome with lapatinib-trastuzumab over trastuzumab. Trial registration NCT00429299.

Background Despite endocrine therapy being the mainstay of treatment for hormone receptor positive (HR+)/HER2− metastatic breast cancer, patients at risk of visceral crisis or doubt for endocrine sensitivity are still offered first-line chemotherapy. Maintenance hormonal therapy is generally offered at the discontinuation of chemotherapy. The MAINtenance Afinitor study is a randomised, phase III trial comparing maintenance everolimus combined with aromatase inhibitors (AIs) versus AI monotherapy in patients with disease control after first-line chemotherapy. Methods Patients with stable disease, partial response or complete response after first-line chemotherapy were randomised to everolimus plus AIs (exemestane or letrozole or anastrozole) or to AIs alone. Primary aim was progression-free survival (PFS). Secondary aims included response rate, safety and overall survival (OS). Results In total, 110 patients were randomised to everolimus + AIs (n = 52) or to AIs (n = 58). Median PFS was 11.0 months (95% confidence interval [CI] 8.1–13.8) in the everolimus + AI arm and 7.2 months (95% CI 4.7–10.9) in the AI monotherapy arm (hazard ratio [HR] 0.71, 95% CI 0.47–1.06). Objective response rate was 22.4% in everolimus + AI arm and 19.2% in AI monotherapy arm. A higher proportion of disease progression as best response was reported in the AI monotherapy arm (28.8% versus 14.3%). Median OS was 35.7 months (95% CI 26.0–47.8) in the combination arm versus 33.5 (95% CI 26.4–42.7) in the AI alone arm (HR 1.0, 95% CI 0.61–1.62). Conclusions EVE + AIs did not significantly impact on the outcome of metastatic breast cancer patients deemed suitable for first-line chemotherapy. Also taking into account treatment tolerability, maintenance endocrine therapy remains the standard. Trial registration EudraCT: 2013-004153-24.

547 Background: Optimal adjuvant endocrine therapy for HER2+/HR+ patients treated with chemotherapy and trastuzumab is still unclear. We evaluated the impact of the type of endocrine therapy on DFS in patients with HER2+/HR+ breast cancer enrolled in the phase III ShortHER trial. Methods: The Short-HER study randomized 1254 patients with HER2+ early breast cancer to receive 9 weeks vs 1 year of adjuvant trastuzumab combined with anthracycline-taxane chemotherapy. The type of adjuvant endocrine was collected every 6 months during the first 5 years of follow-up and was classified as: aromatase inhibitor (AI), tamoxifen and aromatase inhibitor (TAM-AI, in case of both drugs were administered for at least 1 year each), or tamoxifen (TAM). For premenopausal patients, the use of GnRH analogue was also collected. DFS was calculated from randomization to disease recurrence (locoregional or metastatic), second primary invasive cancer, or death. Results: 853 patients with HR+ BC (ER and/or PgR >10%) were included: 60% postmenopausal, 40% premenopausal. The pattern of endocrine therapy was: 55% AI, 22% TAM, 15% TAM-AI (8% missing data). Among premenopausal patients, 51% received GnRH. At a median follow up of 8.7 years (IQR 7.6-9.0), patients who received AI had a significantly better DFS as compared to patients who received TAM or TAM-AI: 7-yr DFS 87.3% vs 81.7%, log-rank P=0.017 (HR 1.46, 95%CI 1.05-2.03). In multivariate analysis including menopausal status, stage, and treatment arm, the type of endocrine therapy maintained a significant association with DFS (Table). In the subgroup of premenopausal patients, the use of GnRH was associated with numerically improved DFS: 86.6% vs 81.6%, log-rank P=0.168 (HR=0.70, 95%CI 0.43-1.16). Conclusions: In this post-hoc analysis of the ShortHER trial, adjuvant treatment with aromatase inhibitor was independently associated with improved DFS. Subgroup analysis in premenopausal patients suggests potential benefit with ovarian suppression. Clinical trial information: NCT00629278. [Table: see text]

Purpose: We explored the prognostic effect of PIK3CA mutation in HER2+ patients enrolled in the ShortHER trial. Patients and Methods: The ShortHER trial randomized 1,253 patients with HER2+ breast cancer to 9 weeks or 1 year of adjuvant trastuzumab combined with chemotherapy. PIK3CA hotspot mutations in exon 9 and 20 were analyzed by pyrosequencing. Expression of 60 genes, including PAM50 genes was measured using the nCounter platform. Results: A mutation of the PIK3CA gene was detected in 21.7% of the 803 genotyped tumors. At a median follow-up of 7.7 years, 5-year disease-free survival (DFS) rates were 90.6% for PIK3CA mutated and 86.2% for PIK3CA wild-type tumors [HR, 0.84; 95% confidence interval (CI), 0.56–1.27; P = 0.417]. PIK3CA mutation showed a favorable prognostic impact in the PAM50 HER2-enriched subtype (n = 232): 5-year DFS 91.8% versus 76.1% (log-rank P = 0.049; HR, 0.46; 95% CI, 0.21–1.02). HER2-enriched/PIK3CA mutated versus wild-type tumors showed numerically higher tumor-infiltrating lymphocytes (TIL) and significant upregulation of immune-related genes (including CD8A, CD274, PDCD1, and MYBL2, a proliferation gene involved in immune processes). High TILs as well as the upregulation of PDCD1 and MYBL2 were associated with a significant DFS improvement within the HER2-enriched subtype (HR, 0.82; 95% CI, 0.68–0.99; P = 0.039 for 10% TILs increment; HR, 0.81; 95% CI, 0.65–0.99; P = 0.049 for PDCD1 expression; HR, 0.72; 95% CI, 0.53–0.99; P = 0.042 for MYBL2 expression). Conclusions: PIK3CA mutation showed no prognostic impact in the ShortHER trial. Within the HER2-enriched molecular subtype, patients with PIK3CA mutated tumors showed better DFS versus PIK3CA wild-type, which may be partly explained by upregulation of immune-related genes.

This abstract was withdrawn by the authors.

Background Although human epidermal growth factor receptor 2 (HER2) overexpression is associated with poor prognosis, patients (pts) with pT1a N0M0 breast cancers (BCs) have an excellent outcome across all subtypes. Interval cancers (ICs) have poorer survival than screen-detected (SD) tumours, and an association has been reported between ICs and HER2 overexpression. We aimed to determine, in a general population of pT1a N0M0 BCs with known screening status, whether HER2-positive ICs have a poorer outcome than HER2-positive SD cancers. Methods We evaluated all incident pT1a N0M0 BCs (n = 874) collected in the Emilia-Romagna region (Italy) from 2003 to 2009 and diagnosed in women aged 50–69. Pts unexposed to screening, with unknown HER2 status and/or treated with adjuvant trastuzumab were excluded from analysis. Results Sixty-one percent of the BCs were SD, whereas 19% were ICs. BCs with high histologic grade, hormone receptor–negative or HER2-positive status (odds ratio = 1.7; 95% confidence interval [CI]: 1.1–2.7) were more likely ICs. Median follow-up was 115 months. The 10-year invasive disease-free survival (iDFS) for HER2-positive ICs was lower than that for HER2-positive SD cancers: 75.0% (95% CI: 55.5%–94.5%) versus 93.8% (95% CI: 86.5%–100%). An interaction between ICs and HER2-positive status was found for poorer iDFS after adjusting for prognostic variables (HR = 5.3; 95% CI: 1.6–16.7). Conclusions IC detection may identify pts with HER2-positive pT1a N0M0 tumours in whom the rate of recurrence justifies consideration for conventional, anti-HER2, adjuvant treatment.

PURPOSE: The aim was to evaluate the role of tumor-infiltrating lymphocytes (TIL) in predicting molecular response after preoperative endocrine or cytotoxic treatment for HR+/HER2- patients who do not achieve a pathological complete response. METHODS: Stromal (Str) TIL were centrally evaluated on samples from diagnostic core-biopsies of HR+/HER2- patients included in two prospective randomized trials: the LETLOB trial (neoadjuvant endocrine-based treatment) and the GIOB trial (neoadjuvant chemotherapy-based treatment). Pre- and post-treatment Ki67 was centrally assessed. RESULTS: StrTIL were evaluable in 111 cases (n = 73 from the LETLOB trial and n = 38 from the GIOB trial). Median StrTIL was 2%. Patients with high StrTIL (StrTIL ≥10%, n = 28) had more frequently breast cancer of ductal histology (p = 0.02), high grade (p = 0.049), and high Ki67 (p = 0.02). After neoadjuvant endocrine treatment (LETLOB cohort), a significant Ki67 suppression (p < 0.01) from pre- to post-treatment was observed in both the low and high StrTIL groups. High StrTIL patients achieve more frequently a relative Ki67 suppression ≥50% from baseline as compared to low StrTIL patients (55 vs. 35%, p non significant). After neoadjuvant chemotherapy (GIOB cohort), a significant Ki67 suppression was observed only for low StrTIL patients (Wilcoxon p = 0.001) and not in the high StrTIL group (p = 0.612). In this cohort, the rate of patients achieving a relative Ki67 suppression ≥50% from baseline was significantly higher in the low vs high StrTIL group (64% vs 10%, p = 0.003). Geometric mean Ki67 suppression was evaluated in each cohort according to StrTIL: the lowest value (-41%) was observed for high StrTIL cases treated with chemotherapy. CONCLUSIONS: This hypothesis-generating study suggests that in HR+/HER2- breast cancer StrTIL at baseline may influence the achievement of a molecular response after neoadjuvant treatment. Further evaluation in large studies is needed, and interaction with the type of treatment warrants to be explored.

Background: In metastatic breast cancer, the CDK4,6 inhibitor palbociclib associated with fulvestrant proved superior to fulvestrant alone in HER2-negative breast cancer (NEJM 2015). In HER2-positive tumors, the dual blockade of the receptor plus an aromatase inhibitor led to a 21% rate of pCR in a neoadjuvant setting in ER+ cancers (J Clin Oncol 2013). In preclinical studies concomitant inhibition of CDK4,6 and trastuzumab led to synergistic antitumor activity (Cancer Cell 2016). The extent of early change and the persistence of Ki67 down-regulation are robust markers of the effects of endocrine treatments in hormone receptors positive breast carcinomas in the setting of neoadjuvant endocrine therapy (Ann Oncol 2012). Methods: In this exploratory Phase II trial (NCT02530424), women with invasive unilateral non metastatic ER-positive breast cancer expressing HER2 and suitable for neoadjuvant therapy were treated with every 3 wks trastuzumab and pertuzumab for 6 cycles combined with palbociclib 125 mg po q.d. x 21 q. 4 wks and fulvestrant i.m. 500 mg, both given for 5 cycles (HPPF). The primary endpoint was characterization of Ki67 changes from baseline before therapy, at 2 weeks and at surgery. Results: A total of 23 patients with centrally confirmed HER2 and ER positive breast cancer were recruited for this study. Ki67 was also assessed centrally. At baseline 30% of cases were classified as locally advanced, 43% as cN0 and 78% had Ki67 values > 20%. Objective clinical response was documented in 96% of patients and pCR (absence of invasive cells in breast and axillary nodes) was documented in 22% (pCR in breast only was 26%). The geometric means of Ki67 expression assessed at baseline, after 2 weeks of treatment and at surgery were 30.8 (SD 15.1), 3.9 (SD 15.9) and 9.2 (SD 16.6) respectively. The mean change in Ki67 values from baseline to after 2 weeks was -24.5 (paired t-test: P < 0.0001) and -13.9 (P = 0.008) from baseline to surgery. Treatment was fairly well tolerated. No serious adverse events > grade 3 were reported. The most frequent G 3 adverse events were neutropenia (26% of patients) and gastrointestinal disorders (17%). Conclusions: Neoadjuvant triple targeting of ER, HER2 and Rb in HER2+/ER+ breast cancer treatment caused a significant and rapid decrease of Ki67 that was of larger magnitude after 2 weeks than at surgery irrespective of the recorded objective clinical response. The good tolerability, the rapid effect on Ki67, the consistent clinical response, and the rate of pCR with this chemotherapy-free approach support further clinical testing and additional molecular characterization. Supported in part by an unrestricted grant from Pfizer Italia S.r.l. and from Roche S.p.a. Italia Citation Format: Gianni L, Bisagni G, Colleoni M, Del Mastro L, Zamagni C, Mansutti M, Zambetti M, Frassoldati A, De Fato R, Valagussa P, Viale G. Neo-adjuvant treatment with trastuzumab and pertuzumab associated with palbociclib and fulvestrant in HER2-positive and ER-positive breast cancer: Effect on Ki67 during and after treatment. A phase II Michelangelo study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-39.

Background. Although long considered a tumor suppressor gene, PML (promyelocytic leukemia) also plays tumor-promoting functions in specific contexts. In vitro and in vivo studies have demonstrated that PML is upregulated by HIF-1α transcriptional activation in triple-negative breast cancer (TNBC) cells and it is implicated in promoting metastasis downstream of HIF-1α. This pro-metastatic function of PML is inhibited by arsenic trioxide, a pharmacological compound currently in use in acute promyelocytic leukemia. However, the clinical relevance of PML expression in BC has not been extensively investigated. In this study, we evaluated the association of PML expression with clinic-pathological factors and outcome (pathological complete response -pCR- and risk of recurrence) in the ETNA trial, and risk of recurrence in the TCGA. Methods. In the ETNA study (NCT01822314) 695 patients with HER2-negative breast cancer (BC) were randomized to receive neoadjuvant paclitaxel or nab-paclitaxel followed by 4 cycles of an anthracycline regimen. In the ITT study population, the two treatments did not show significantly different rates of pCR nor different Event-Free Survival (EFS) (Gianni JAMA Oncol 2018, Gianni ASCO 2019). A central histologic assessment of ER, PgR, HER2 status and Ki67 was mandatory. We evaluated PML expression by immunohistochemistry using the continuous histoscore (H-score) on pre-treatment core biopsies. The H-score is generated from the estimation of the percentage of cells with no (0), light (1+), moderate (2+) and strong (3+) intensity staining, and the corresponding score is generated with the following algorithm: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. We evaluated the association of PML with clinic-pathological features and clinical outcomes (pCR and EFS) in triple negative (TN) and in LuminalB-like (ER+ and/or PgR+, Ki67≥14%) groups. We also investigated the association between PML mRNA expression (RNA-seq) and DFS in HER2-negative BC (TN, n=192; ER+/HER2-, n=702) in the TCGA dataset. Results. In the ETNA study, PML was successfully assessed and valuable in 491 pts (70.6%). The average PML expression was 126.3 (median 120, range 0-295). In the ETNA trial, TNBC showed the highest expression of PML (p20%, LumB-high) (p=0.0005). However, within the LumB-high group, higher proliferation (Ki67>40%) showed a higher expression of PML (p=0.025), suggesting a non-linear relationship between PML and proliferation in luminal tumors. In LumB-high, PML was higher in PgR-negative tumors (p=1.0E-5). Finally, PML showed a positive association with higher stromal tumor-infiltrating lymphocytes (sTILs) both in LumB-like and TN group (p=0.019 and p=0.001, respectively). PML expression was not significantly associated with pCR and risk of recurrence in LumB-like nor in TN BC.In the TCGA dataset also PML expression was highest in the TN group (p Citation Format: Stefania Zambelli, Chanel Smart, Giampaolo Bianchini, Isabella Sassi, Mauro Mansutti, Antonio Anton, Lourdes Calvo, Giancarlo Bisagni, Begona Bermejo, Martina Uggè, Barbara Galbardi, Vladimir Semiglazov, Marc Thill, Jose Ignacio Chacon, Arlene Chan, Serafin Morales Murillo, Isabel Alvarez, Ainhara Lahuerta, Patrizia Zucchinelli, Claudio Doglioni, Pinuccia Valagussa, Ignasi Tusquets, Luca Gianni, Rosa Bernardi. Prognostic and predictive value of PML in the ETNA study and the TCGA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-21.

Background: Tumor-infiltrating lymphocytes (TILs) have been reported to be associated with increased therapeutic efficacy of trastuzumab/pertuzumab-based neoadjuvant therapy (NT) in patients (pts) with HER2-positive breast cancer (BC). Subcutaneous (SC) trastuzumab has non-inferior efficacy to intravenous (IV) administration, with a similar safety profile. Interestingly, SC trastuzumab has been observed to be more immunogenic than IV trastuzumab and act at different immunologic levels. Therefore, by modifying the modality of administration of trastuzumab, it could be possible to interfere with different immune pathways and exert a favorable immunomodulation in HER2-positive BC. Methods: In this non-comparative, phase II, neoadjuvant, randomized study, pts were eligible if they had previously untreated, histologically confirmed, locally advanced, inflammatory, or early-stage HER2-positive BC. Pts were treated with FEC (fluorouracil 500 mg/m2; epirubicin 75 mg/m2; cyclophosphamide 500 mg/m2) q21 × 3 cycles. Then, they were randomly assigned (1:1) to receive: docetaxel (75 mg/m2) plus pertuzumab (840 mg loading dose (LD), then 420 mg) plus IV trastuzumab (8 mg/kg LD, then 6 mg/kg) q21 × 4 cycles (arm A) or, docetaxel plus pertuzumab plus SC trastuzumab (fixed dose of 600 mg) q21 × 4 cycles (arm B). After surgery, pts received trastuzumab q21 × 14 cycles using the same formulation (SC or IV) of the preoperative phase. The primary endpoint was the rate of stromal TILs (sTILs) on residual disease after surgery. Tumor biopsy and posttreatment surgical samples were centrally analyzed for TILs. Blood samples were also collected during NT for tumor-specific lymphocyte cell activity analysis. Feasibility, efficacy and safety were also evaluated. ClinicalTrials.gov: NCT03144947. Results: Between November 2016 and September 2017, according to an adaptive Simon9s two-stage optimal design, we enrolled 65 pts, of whom two were deemed ineligible for the study. Thus, 63 pts (31 in arm A and 32 in arm B) were assessed for the primary and secondary endpoints. The pathologic complete response (pCR; no invasive tumor in breast and axilla) rates were 64.5% (95% CI, 47-81) in arm A, and 59.4% (95% CI, 42-76) in arm B. The most common adverse events of grade 3 or higher were neutropenia (15 [48.4%] pts in arm A, and 11 [34.4%] in arm B), neurotoxicity (1 [3.2%], and 2 [6.2%], respectively), and diarrhea (1 [3.2%], and 1 [3.1%], respectively). There were no events of congestive heart failure. At surgery, 11 pts in arm A and 13 pts in arm B were evaluable for TIL analysis. The median value of sTILs (7.5%) on pre-treatment tumor biopsies was used as the cut-off value, and high sTIL levels were observed in 27.3% and in 46.1% of residual tumors after treatment arm A and B, respectively. There was a positive correlation between pretreatment sTILs and PD-L1 expression on stromal immune cells (Kendall’s τ =0.80). Interestingly, a significant inverse correlation was observed between PD-L1 expression on pretreatment sTILs and the T cell co-receptor CD3 expressed on posttreatment sTILs (Pearson’s ρ = -0.70). This finding was particularly evident in the arm B group (ρ = -0.85). Conclusions: NT with either SC or IV trastuzumab in combination with pertuzumab and chemotherapy had a significant effect on sTIL expression at surgery. In particular, the SC trastuzumab-based arm exerted the most relevant enrichment of sTILS in posttreatment residual tumors. These findings suggest a role for the SC administration of trastuzumab in determining favorable variations of host immune response parameters among pts with HER2-positive early BC who had residual disease after NT. Citation Format: Antonino Musolino, Stefania Gori, Elisabetta Cretella, Alessandra Marabese, Luigi Cavanna, Antonio Frassoldati, Giancarlo Bisagni, Chiara Casarini, Emilio Bria, Luisa Carbognin, Elena Fiorio, Alba A Brandes, Claudio Zamagni, Lorenzo Gianni, Alberto Zambelli, Filippo Montemurro, Michele Tognetto, Renata Todeschini, Giuseppe Maglietta, Gabriele Missale, Enrico M Silini. Phase II, open label, randomized, biomarker study of immune-mediated mechanism of action of neoadjuvant subcutaneous trastuzumab in patients with operable, locally advanced, or inflammatory HER2-positive breast cancer. ImmunHER trial on behalf of the Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-19.

Dose-dense adjuvant chemotherapy is standard of care in high-risk early breast cancer patients. However, its role in HER2-positive patients is still uncertain. In this exploratory analysis of the GIM2 trial, we investigated the efficacy of dose-dense chemotherapy in HER2-positive breast cancer patients with or without exposure to trastuzumab. In the GIM2 trial, node-positive early breast cancer patients were randomized to receive 4 cycles of (fluorouracil)epirubicin/cyclophosphamide followed by 4 cycles of paclitaxel administered every 2 (dose-dense) or 3 (standard-interval) weeks. After approval of adjuvant trastuzumab, protocol was amended in April 2006 to allow use of trastuzumab for 1 year after chemotherapy completion in HER2-positive patients. The efficacy of dose-dense chemotherapy in terms of disease-free survival (DFS) and overall survival (OS) was assessed according to HER2 status and trastuzumab use. Out of 2,003 breast cancer patients, HER2 status was negative/unknown in 1,551 patients; among the 452 patients with HER2-positive breast cancer, chemotherapy alone or followed by trastuzumab was given to 320 and 132 patients, respectively. Median follow-up was 8.1 years. No significant interaction between HER2 status, trastuzumab use and chemotherapy treatment was observed for both DFS (p=0.698) and OS (p=0.708). Nevertheless, there was no apparent benefit in the HER2-positive group treated with trastuzumab (DFS: HR, 0.99; 95% CI 0.52-1.89; OS: HR, 0.95; 95% CI 0.37-2.41). Although dose-dense chemotherapy was associated with a significant survival improvement in high-risk breast cancer patients, its benefit appeared to be smaller (if any) in patients with HER2-positive disease who received adjuvant trastuzumab. This article is protected by copyright. All rights reserved.

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

Background Considering the high proportion of tumor-infiltrating lymphocytes (TILs) in BRCA-related breast cancer, we expect that PD-1 pathway is highly expressed and PD-1 antagonist pembrolizumab could provide clinical activity in this kind of tumor. Furthermore, BRCA-related breast cancers are known to be more sensitive to platinum-derived drugs. Thus the association between Pembrolizumab and Carboplatin in metastatic BRCA-related breast cancer seems to be active in this setting of patients. This study will evaluate the safety and the efficacy of Pembrolizumab associated with Carboplatin in BRCA mutated or with unknown mutations metastatic breast cancer patients. Study and Statistical DesignThis is a national multicenter two-stage single arm phase II study, enrolling BRCA mutated or with unknown mutations metastatic breast cancer patients. The sample size has been estimated by using the two-stage Simon’s design. In the first stage, 20 subjects will be enrolled. If, after first stage ≤11 responses (r1) will be observed, accrual will terminate and the experimental regimen will be rejected. Otherwise if 12 or more responses will be seen then the accrual will continue to the second stage of an additional 33 subjects (total, 53 subjects). At the second stage if 33 or less responses out of 53 subjects will be observed the treatment will be rejected. With the null hypothesis (p0) being equal 0.55 with a type I (alfa) error of 0.10 (10%) and a type II (beta) error of 0.20 (power=80%) and the alternative hypothesis (p1) is 0.70, (response rate 70%), we expect to reach an overall response rate (ORR) ≥ 70% by the combination of Pembrolizumab plus Carboplatin. We also expect to reach a median Time to Progression (TTP) and Overall Survival (OS) of five and fifteen months respectively. The Disease Control Rate (DCR) will be expected as ≥ 80% by the combination of Pembrolizumab plus Carboplatin Study TreatmentCarboplatin at area under the time-concentration curve 6 (AUC 6) intravenously once every 3 weeks in combination with Pembrolizumab 200 mg intravenously every 3 weeks will be administered for six courses and then only Pembrolizumab alone will continue until occurrence of unacceptable toxicities or disease progression. Eligibility CriteriaIn order to be eligible for participation in this trial, the subject, aged ≥ 18 years, must have metastatic confirmed breast cancer, with a disease progression by radiological techniques within 12 months prior to signing informed consent, and a documented mutation in BRCA1 or BRCA2 genes that is predicted to be deleterious or suspected deleterious or with unknown significance. The subject must have measurable disease based on RECIST 1.1 and have a performance status of 0 or 1 on the Eastern Cooperative Oncology Group (ECOG) Performance Scale. Prior chemotherapy with anthracyclines and taxanes has to be administered in neoadjuvant or adjuvant setting. In case of luminal tumors hormonal treatments for advanced disease can be administered before. The life expectancy must be greater than 3 months and the subject must demonstrate adequate organ function by screening labs performed within 10 days of treatment initiation. Objectives and HypothesisThe primary end-point will be the ORR, evaluated according to RECIST criteria. Secondary objectives will be the TTP, the duration of response (DOR ), the DCR, and the OS. The safety of the combination will be evaluated according to the worst toxicity grade reported throughout the whole treatment period.The Exploratory Objective will be the evaluation of ORR, TTP, DOR, and DCR based on irRECIST. Biological parameters of CD8/TILs and PD-L1 will be considered in the metastatic biopsy. Target AccrualThe first subject was enrolled in January 2019 and recruitment is ongoing. Enrollment of the first 20 subjects is expected to complete in Q2 2021. Citation Format: Laura Cortesi, Marta Venturelli, Federica Caggia, Luigi Marcheselli, Annita Gozzi, Alberto Zambelli, Valentina Guarneri, Antonino Musolino, Elena Fiorio, Giancarlo Bisagni, Andrea Rocca, Valentina Arcangeli, Elisabetta De Matteis, Sergio Rizzo, Andrea Michelotti. A phase II study to evaluate the efficacy and safety of pembrolizumab plus carboplatin in BRCA-related metastatic breast cancer: PEMBRACA trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-31-01.

Background: We previously reported the correlation between tumor infiltrating lymphocytes (TIL) at baseline and pathological complete response (pCR) after neoadjuvant chemotherapy plus anti-HER2 agents for HER2-positive patients included in the prospective randomized CherLOB study (Dieci , SABCS 2014). Here we report the survival analysis results. Methods: The phase II neoadjuvant CherLOB study (Guarneri, J Clin Oncol 2012) randomized 121 HER2-positive, stage II-IIIA breast cancer patients to anthracyclines/taxane-based chemotherapy plus trastuzumab, lapatinib, or both. A part from endocrine therapy for hormone receptor-positive patients, adjuvant treatment was left to physicians' decision. This included trastuzumab for up to 1 year. Intratumoral (It) and stromal (Str) TIL were centrally evaluated on Hematoxylin and eosin-stained slides from pre-treatment biopsies and post-treatment surgical samples. For the present analysis, follow up was updated as of May 2015 (median follow up 68.3 months). Event-free survival (EFS) was calculated as the time interval from randomization to: recurrence, second primary cancer, death from any cause. Results: Among the 118 patients evaluable for pathological response, pCR (ypT0/is ypN0) was confirmed as a strong prognostic factor for EFS (HR 0.15, 95% CI 0.04-0.64, p=0.01). Both pre-treatment ItTIL and StrTIL evaluations were available for 105 patients, median values were 5% and 17% (interquartile range 0%-15% and 9%-40%, respectively). Table 1 summarizes the results of the survival analysis for the association of TIL with EFS. Table 1Continuous TILneventsHR95%CIpItTIL 10% increments105180.490.23-1.10.068StrTIL 10% increments105180.820.64-1.050.11Categorical TILneventsHR95%CILog-rank pItTIL above median; ItTIL below median51;545;130.390.14-1.090.061StrTIL above median; StrTIL below median52;538;100.750.3-1.90.61 A non-significant trend for better EFS with increasing It-TIL (per 10%) was observed. Patients with ItTIL above the median value had a more prolonged EFS compared to patients with ItTIL below median (5-yrs EFS rate 89% vs 76%), with borderline statistical significance for the comparison between the two groups. No difference according to StrTIL above vs below median value was observed (5-yrs EFS rate 83% vs 82%, respectively). A comparison between lymphocyte predominant (LP) tumors, as defined by the generally accepted cut-off of ItTIL and/or StrTIL >=60%, vs non-LP cases was not performed due to low number of patients (n=17) and events (n=1) in the LP group. Among the 63 patients with residual disease, ItTIL and StrTIL evaluated on the surgical sample were not associated to EFS. Conclusions: In this low-powered analysis, TIL did not provide significant prognostic information for HER2-positive breast cancer patients treated in the CherLOB study. However, a non-significant trend suggests a positive correlation between increased levels of It-TIL and better EFS. The evaluation of immune gene expression signatures and their correlation with survival is ongoing, results will be available for the meeting. Citation Format: Dieci MV, Bisagni G, Cagossi K, Generali DG, Sarti S, Piacentini F, Conte P, Guarneri V. Survival analysis of the prospective randomized Cher-Lob study: Correlation with tumor infiltrating lymphocytes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-03.

TPS598 Background: Chemotherapy represents, today, the only treatment option for triple negative breast cancer (TNBC) and still a considerable proportion of pts with primary TNBC experience disease relapse. The risk is particularly high in the presence of poor prognostic features, such as more advanced stage and, for pts treated with neoadjuvant chemotherapy, failure to achieve a pCR. Recent evidence suggest that immunotherapy can play a major role in TNBC pts. Methods: The A-BRAVE trial is an investigator-driven trial sponsored by the University of Padova (Dept. of Surgery, Oncology and Gastroenterology). This is a phase III, multicentric, randomized adjuvant study that compares 1 year of treatment with the anti PD-L1 avelumab vs observation for pts who completed treatment with radical intent for primary TNBC including surgery and chemotherapy. The study enrolls pts in two strata: -Stratum A: primary TNBC pts who completed surgery followed by adjuvant, defined according to one of the following stage categories: if pN2, any pT; if pN1, pT > 2 cm; if pN0, pT > 5 cm. -Stratum B: primary TNBC pts who completed neoadjuvant chemotherapy followed by surgery who did not achieve pCR. Pts who also received additional adjuvant chemotherapy for no more than 6 months are eligible in Stratum B, after the completion of the adjuvant chemotherapy. Pts are randomized (1:1, balanced for strata A and B) to receive Avelumab 10 mg/kg I.V. q2w for 1 year or to observation. The first and second co-primary endpoints are disease-free survival (DFS) in all pts and DFS in Stratum B pts. With a planned sample size of n = 474 pts the trial has 90% power to detect a HR = 0.60 for the first co-primary endpoint (n = 172 events required). Taking into account that the percentage of patients enrolled in the stratum B could range from 70 to 80%, there will be 70-79% power to detect a HR = 0.60 at alpha allocated in this patient subgroup (second co-primary endpoint). Secondary objectives include: DFS in PD-L1 positive pts, overall survival, safety, biomarkers. Tumor tissue, plasma samples and fecal samples are collected for biomarker analysis. The study is currently recruiting across 73 sites in Italy and UK. As of February 2020, n = 349 pts have been enrolled. EUDRACT 2016‐000189‐45. The authors present the A-BRAVE trial in progress on behalf of Italian and UK investigators. Clinical trial information: NCT02926196 .

582 Background: The CHER-LOB randomized phase II study showed that the combination of lapatinib and trastuzumab plus chemotherapy increases the pathologic complete response (pCR) rate compared with chemotherapy plus either trastuzumab or lapatinib. Here we report the results of survival analysis according to treatment arm and pCR. Methods: The CherLOB study randomized 121 HER2-positive, stage II-IIIA breast cancer patients to anthracyclines/taxane-based chemotherapy plus trastuzumab, lapatinib, or both. After surgery, patients received adjuvant trastuzumab for up to 1 year. The primary end point of the study was met, with a relative increase of 80% in the pCR rate achieved with chemotherapy plus trastuzumab and lapatinib compared with chemotherapy plus either trastuzumab or lapatinib (Guarneri, J Clin Oncol 2012). Relapse-free survival (RFS) was calculated from randomization to breast cancer recurrence (locoregional or distant) or death from any cause, whichever first. Overall survival (OS) was calculated from randomization to death from any cause. Results: At a median follow up of 8.8 years, RFS rates at 5 years were: 85.8% in the trastuzumab + lapatinib arm, 77.8% in the trastuzumab arm, 78.1% in the lapatinib arm (log-rank p = 0.160). Patients treated with dual HER2 blockade (trastuzumab + lapatinib arm) experienced numerically better RFS as compared to patients treated with single HER2 blockade (trastuzumab arm and lapatinib arm combined): 5-yr RFS 85.8% vs 78.0%, log-rank p = 0.087; HR = 0.51, 95% CI 0.23-1.12, p = 0.093. The achievement of pCR was a strong prognostic factor. 5-yr RFS rate was 97.3% for pCR patients vs 72.9% for non-pCR patients (log-rank p < 0.001, HR = 0.12, 95% CI 0.03-0.49, p = 0.003); similar significant results were observed in both the estrogen receptor-negative and estrogen-receptor positive subgroups. OS was also improved in pCR patients: 8-yr OS rates were 97.2% vs 80.0% for non pCR patients (log-rank p = 0.028, HR = 0.14, 95% CI 0.02-1.08, p = 0.060). Conclusions: In the Cher-LOB study, there was a not statistically significant signal for a better RFS for patients who received dual HER2 blockade with trastuzumab and lapatinib plus chemotherapy as compared to patients treated with single anti-HER2 agent (trastuzumab or lapatinib) plus chemotherapy. Patients achieving a pCR had longer RFS and OS as compared to non-pCR patients. Clinical trial information: NCT00429299 .

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

Background. We investigated the association between TILs assessed by different metrics and clinical outcome (pathological complete response -pCR- and risk of recurrence) in the ETNA trial. Methods. In the ETNA study (NCT01822314) 695 patients with HER2-negative breast cancer (BC) were randomized to receive neoadjuvant paclitaxel or nab-paclitaxel followed by 4 cycles of an anthracycline regimen. In the ITT study population, the two treatments did not show significantly different rates of pCR nor different Event-Free Survival (EFS) (Gianni JAMA Oncol 2018, Gianni ASCO 2019). We measured TILs by optical evaluation on pre-treatment core biopsies using three different metrics: stromal and intratumoral TILs (sTILs, iTILs), and hotspot TILs (hTILs)ie. the field of highest TILs density, in order to capture some of the spatial heterogeneity of TILs reported as more informative in ER+ BCs (Heindl JNCI 2018). We then investigated the association of TILs with pCR and EFS separately in TN and in LuminalB-like (ER+ and/or PgR+, Ki67≥14%) groups, as well as possible treatment-biomarker interactions. Results. TILs could be assessed in 589 pts (85%). The three TILs metrics were significantly correlated (p20%, n=126) TILs. In the low hTILs group, abraxane was significantly more effective than paclitaxel (HR 0.64 [0.26-0.89], p=0.02). In the high hTILs group, paclitaxel was associated with better long-term outcome (HR 3.79 [1.33-10.8], p=0.01). If the same cut-off was applied in TN BC, abraxane had a numerical trend for superiority compared to paclitaxel in the low hTILs tumors (HR 0.64 [0.34-1.20]). No differences were observed in the high TIL group. Conclusions. Pre-treatment assessment of TILs in the ETNA trial was predictive of the likelihood of pCR regardless of the tumor subtype, and was prognostic in the TN subgroup. The assessment of hotspot TILs seems to be more informative than average stromal TILs in LumB-like tumors. Finally, different levels of hTILs at baseline were associated with different benefit from abraxane compared to paclitaxel. This finding warrants independent confirmation, and the mechanism for the association is being investigated. Supported in part by an unrestricted grant from Celgene Sarl, Swizerland Citation Format: Giampaolo Bianchini, Chanel Smart, Mauro Mansutti, Antonio Anton, Luca Licata, Isabella Sassi, Lourdes Calvo, Giancarlo Bisagni, Begona Bermejo, Vladimir Semiglazov, Marc Thill, Jose Ignacio Chacon, Arlene Chan, Serafin Morales Murillo, Isabel Alvarez, Ainhara Lahuerta, Patrizia Zucchinelli, Claudio Doglioni, Giuseppe Viale, Pinuccia Valagussa, Ignasi Tusquet, Luca Gianni. Clinical implication of tumor infiltrating lymphocytes (TILs) in the ETNA study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-05.

Background Subcutaneous trastuzumab (H SC) is a valuable alternative to the intravenous formulation. This study assessed H SC safety and tolerability in human epidermal growth factor receptor 2 (HER2)+ early/locally advanced breast cancer (EBC/LABC). Methods SCHEARLY is a prospective, two-cohort, non-randomised, multicentre Italian trial included in the umbrella study UmbHER1, planning a 1-year treatment with H SC 600 mg in HER2+ EBC/LABC. Patients were sequentially assigned to cohort A (N = 121) and B (N = 119) to receive H SC via a handheld syringe and a single-use injection device, respectively. Adjuvant or neoadjuvant treatment included anthracycline-containing regimens followed by H SC plus taxanes and then alone for 18 cycles totally. Results Two hundred forty patients were enrolled (adjuvant therapy: 81.7%; neoadjuvant therapy: 18.3%), and 201 completed the treatment (early discontinuation was mainly due to intercurrent adverse events [AEs], 7.5%). Overall, the two cohorts displayed similar safety profiles. From H SC start, the rate of treatment-related AEs in the safety population (N = 228) was 3.9% for grade ≥3 AEs; 0.9% for serious AEs (one pleuropericarditis and one anaphylactic shock, both resolved) and 14.5% for cardiac AEs, the most common being the decreased left ventricular ejection fraction (7.9%; mean reduction from the screening to the follow-up visit was 2.9%). No cases of congestive heart failure occurred. The rate of systemic administration-related reactions and local injection site reactions was 68.0% and 21.9%, respectively, mostly of grade 1–2. Conclusions H SC 600 mg confirmed to be a safe and tolerable option as adjuvant/neoadjuvant therapy in patients with HER2+ EBC and LABC. ClinicalTrials.gov Identifier NCT01940497.

Background. The management of small (≤ 1 cm), node-negative, HER2+ve BC is controversial, since data from randomized clinical trials specifically addressing the benefit of adjuvant systemic treatment with or without Trastuzumab in this setting are still lacking. The aims of this retrospective study are to assess how pts are managed in routinary clinical practice in Italy, whether clinical or biological features may influence the choice of adjuvant systemic therapy and if there is any difference in the outcome between treated and not treated pts. Patients and methods. Data of 268 consecutive pts who underwent surgery from January 2007 to December 2012 for HER2+ve, pT1a-b pN0 BC, were collected from 25 Italian centres. Descriptive statistical analyses and multivariate logistic regression models were used, with the aim of investigating the relationship between the baseline clinical and biological features and the adjuvant treatment strategy. Results. Pts characteristics were: median age 57, 69% postmenopausal status, 77% had conservative surgery, 32% pT1a, 68% pT1b, 48% G3, 66% ER+ve, 75% Ki67 ≥14%. Ninety percent of pts received adjuvant systemic therapy: 19% hormone therapy (HT) alone, 3% chemotherapy (CT) +/- HT, 64% Trastuzumab + CT +/- HT and 4% Trastuzumab + HT. At the multivariate analysis, the odds of being treated with adjuvant systemic therapy with or without Trastuzumab, resulted higher in presence of conservative surgery (p=0.002), pT1b (p Conclusion. This preliminary analysis shows that in Italy the majority of these pts received systemic adjuvant treatment and about 2/3 were treated with Trastuzumab. Pathological tumor size (pT1b) and negative hormone receptor status represent the main factors influencing the choice of including Trastuzumab in the adjuvant treatment. Survival data are still not mature to drive definitive conclusions about outcome. Citation Format: Stefania Gori, Monica Turazza, Simona Duranti, Elena Fiorio, Jennifer Foglietta, Marcella Gulisano, Ilaria Marcon, Marta Gubbiotti, Maria Giovanna Cavazzini, Simon Spazzapan, Valeria De Simone, Giancarlo Bisagni, Chiara Saggia, Luigi Cavanna, Emilio Bria, Laura Iezzi, Elisabetta Cretella, Patrizia Vici, Daniele Santini, Alessandra Fabi, Ornella Garrone, Antonella Ferro, Silvana Saracchini, Lucia Evangelisti, Sandro Barni, Lucia Mentuccia, Lucio Laudadio, Alessandro Inno, Gianluigi Lunardi, Francesca Coati, Luca Boni. The Promher Study: An observational Italian study on HER2+ve, pT1a-b, pN0, M0 breast cancer (BC) patients (pts) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-18-05.

Background The combination of the dual HER1/HER2 inhibitor lapatinib (L) and capecitabine (C) is a therapeutic option for patients (pts) with HER2+MBC whose disease progresses after treatment with the monoclonal antibody trastuzumab. At present time, no clinical or pathological factors except HER2 status are clearly recognized as predictors of the activity of LC. We conducted a retrospective analysis of pts with HER2-positive metastatic breast cancer receiving LC after trastuzumab failure to identify factors associated with resistance to LC. Materials and methods We collected clinical and pathological data from 151 pts with HER2+ MBC receiving LC after failing a prior trastuzumab-based treatment (either adjuvant or for metastatic disease) treated at 13 Italian Institutions between March 2007 and December 2013. Time to progression (TTP) and overall survival (OS), calculated by the Kaplan Meier (KM) method, were from LC treatment beginning to disease progression or to death in the absence of progression (TTP), and to the date of death or to the date of last follow-up (OS), respectively. LC resistance was defined as TTP from treatment initiation lower or equal to the median TTP for the overall population. KM curves were compared by the Log-rank test. Logistic regression analysis was used to study predictors of TTP below the median value for patients receiving LC. Analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL). Results At a median follow-up of 41 months (IQR 23-62), median TTP to LC therapy was 7 months (IQR 5.5-8.5) and median OS was 18 months (IQR 10-28). Fifteen pts were excluded because of short follow-up (i.e. on LC treatment and Conclusions A short time to progression during capecitabine and lapatinib (LC-R) is associated with reduced OS in patients failing prior trastuzumab based therapy for HER2+ MBC. Patients who had modest clinical benefit from previous trastuzumab-based therapy could experience LC-R indicating the possibility of primary resistance to anti HER2-treatment. For these patients, alternative targeting strategies are urgently needed. Citation Format: Stefania Gori, Valentina Rossi, Monica Turazza, Elena Fiorio, Alessandra Fabi, Giancarlo Bisagni, Jennifer Foglietta, Daniele Santini, Ida Pavese, Alberto Zambelli, Patrizia Vici, Vita Leonardi, Sandro Barni, Silvana Saracchini, Giuseppe Bogina, Simona Duranti, Alessandro Inno, Gianluigi Lunardi, Filippo Montemurro. Multi-institutional retrospective analysis of clinical and pathological factors predicting resistance to lapatinib-based therapy in HER2 positive metastatic breast cancer (HER2+ MBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-19-25.

Introduction: In vitro studies have shown that lapatinib enhances the immune-mediated cytotoxicity (ADCC) of trastuzumab. FcγR polymorphisms have been associated with both ADCC and clinical activity of trastuzumab in patients with HER2-positive metastatic breast cancer. There are no data on the relationship between these polymorphisms and the combination of trastuzumab plus lapatinib in the early stage setting. We performed a pharmacogenomics analysis of CHER-LOB, a randomized phase II trial of preoperative chemotherapy plus trastuzumab (arm A), lapatinib (arm B), or both (arm C) in HER2-positive operable breast cancer. Methods: FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms were analyzed on DNA from peripheral blood samples. Pathologic complete response (pCR) of genotyped cases was evaluated by FcγR polymorphism and treatment arm. Results: Genotyping was successfully performed in 73/121 (60%) patients. No deviation from the Hardy-Weinberg equilibrium was observed. Similarly to the overall results of the CHER-LOB study, in the subset of patients genotyped in this analysis, a significant improvement in pCR rate was observed in favor of the combination of lapatinib plus trastuzumab (arm C) compared to arm A (OR=3.66, P=0.037), and B (OR=3.03, P=0.049). Such improvement was restricted to carriers of FcγRIIIa V allele (C vs. A, OR=5.33, P=0.043; C vs. B, OR=6.50, P=0.012), while it was not observed in patients with FcγRIIIa F/F genotype (C vs. A, OR=2.14, P=0.642; C vs. B, OR=0.71, P=0.737). Disease free survival (DFS) was not different by treatment arm in all genotyped cases, but a trend toward significance for an interaction between FcγRIIIa V allele and better DFS with the combination of lapatinib plus trastuzumab was detected (P=0.058). No significant associations were observed by FcγRIIa polymorphism. Conclusions: Host-related immune signatures may mediate lapatinib enhanced trastuzumab-dependent ADCC. FcγRIIIa genotypes may help predict different outcomes to lapatinib plus trastuzumab in HER2-Positive Early Breast Cancer. Citation Format: Antonino Musolino, Valentina Guarneri, Nadia Naldi, Beatrice Bortesi, Daniela Boggiani, Paolo Sgargi, Daniele G Generali, Federico Piacentini, Maria V Dieci, Massimo Ambroggi, Katia Cagossi, Lorenzo Gianni, Samanta Sarti, Giancarlo Bisagni, Antonio Frassoldati, Pierfranco Conte, Andrea Ardizzoni. Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of preoperative chemotherapy plus trastuzumab and lapatinib in patients with HER2-positive operable breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-23.

Background No evidence exists to recommend a specific chemotherapy regimen in young breast cancer patients. We performed a pooled analysis of two randomised clinical trials to evaluate the efficacy of adjuvant dose-dense chemotherapy in premenopausal breast cancer patients and its impact on the risk of treatment-induced amenorrhoea. Patients and methods In the MIG1 study, node-positive or high-risk node-negative patients were randomised to 6 cycles of fluorouracil/epirubicin/cyclophosphamide every 2 (dose-dense) or 3 (standard-interval) weeks. In the GIM2 study, node-positive patients were randomised to 4 cycles of dose-dense or standard-interval EC or FEC followed by 4 cycles of dose-dense or standard-interval paclitaxel. Using individual patient data, the hazard ratio (HR) for overall survival by means of a Cox proportional hazards model and the odds ratio for treatment-induced amenorrhoea through a logistic regression model were calculated for each study. A meta-analysis of the two studies was performed using the random effect model to compute the parameter estimates. Results A total of 1,549 patients were included. Dose-dense chemotherapy was associated with a significant improved overall survival as compared to standard-interval chemotherapy (HR, 0.71; 95% confidence intervals [CI], 0.54–0.95; p = 0.021). The pooled HRs were 0.78 (95% CI, 0.54–1.12) and 0.65 (95% CI, 0.40–1.06) for patients with hormone receptor-positive and -negative tumours, respectively (interaction p = 0.330). No increased risk of treatment-induced amenorrhoea was observed with dose-dense chemotherapy (odds ratio, 1.00; 95% CI, 0.80–1.25; p = 0.989). Conclusion Dose-dense adjuvant chemotherapy may be considered the preferred treatment option in high-risk premenopausal breast cancer patients.

527 Background: Downregulation of Ki67 by neoadjuvant endocrine therapy predicts activity of endocrine treatments in hormone receptors positive breast cancer. NA-PHER2 is an exploratory phase II study (NCT02530424) assessing Ki67 changes in patients with HER2+ and ER+ breast cancer undergoing dual HER2 block and palbociclib. Cohort A of the NA-PHER2 showed significant decrease of Ki67 at week 2 and at surgery and pathological complete response (pCR) in 27% of patients (Lancet Oncol 2018). Methods: After completing cohort A two additional cohorts were started. In Cohort B cases with HER2 3+ or amplified unilateral breast cancer received therapy with dual block and palbociclib without fulvestrant. In Cohort C tumors with Ki67 >20% and HER2 low (1+/2+, no amplification) received also fulvestrant. Trastuzumab and pertuzumab q3 wks were dosed for 6 cycles and palbociclib for 5 cycles (125 mg po q.d. 3q4 wks). Fulvestrant in Cohort C was given im 500 mg q4 wks for 5 cycles. Primary endpoint was Ki67 change from baseline to 2 weeks and at surgery. Results: 26 eligible patients in cohort B and 23 in cohort C with centrally confirmed HER2 and ER status were recruited. Ki67 was centrally assessed. Main results are reported in the table. Clinical trial information: NCT02530424. The most frequent G >=3 adverse events were neutropenia (36%) and gastrointestinal disorders (12%). Conclusions: Dual block of HER2 and palbociclib caused robust persistent decrease of Ki67 as in cohort A. In cohort B without endocrine therapy there also were pCR and high objective response rate. Effects on Ki67 and ORR were similar in HER2 low tumors. The chemo-free approach of NA-PHER2 leads to promising therapeutic effects and deserves investigation in ER+ HER2+ tumors to spare the toxicity of chemotherapy, and in HER2-low tumors, in which functional activation of HER2 may lead to resistance to endocrine therapy. Supported in part by unrestricted grants of Pfizer Italia S.r.l. and Roche S.p.a. Italia.[Table: see text]

504 Background: The effect of extended adjuvant endocrine therapy (ET) with aromatase inhibitors (AI) after sequential ET with tamoxifen (Tam) followed by AI for 5 years is still controversial. We conduct a clinical trial to assess different durations of ET of letrozole after tam. Methods: The GIM4 LEAD (Gruppo Italiano Mammella 4- Letrozole adjuvant therapy duration study, ClinicalTrials.gov:NCT01064635 ) was a prospective, randomized, Italian multicentric trial. Post-menopausal patients (pts) with hormone receptor positive early breast cancer free of recurrence after 2-3 years of adjuvant tam, were randomized in a 1:1 ratio to receive 3-2 years (short arm, S) or 5 years (long arm, L) of letrozole. The primary study end point was disease-free survival (DFS). Results: Between August 2005 and May 2010, 2056 pts were randomly assigned to receive 3-2 years (n=1030) or 5 years (n=1026) of letrozole. Main patients characteristics in the S and L arms were, respectively: median age 60 vs 61 years, node negative 56 vs 56%, (neo)adjuvant chemotherapy 53.4 vs 54.1%. The median follow-up was 10 years (IQR range: 8.6-11.4). The 8-year DFS was 80% (95% CI:77.3-82.7) and 85% (95% CI:82.9-87.6) in the S and L arm, respectively (hazard ratio, HR 0.82; 95% CI:0.68-0.98; p=0.031). This effect did not change in a multivariate Cox model that included nodal status, grading and age. No evidence of interaction between random assignment and nodal status, age and grading was observed. Among 1960 pts evaluable for toxicity, osteoporosis was diagnosed in 47 (4.8%) in S arm and 81 (8.3%) pts in L arm (chi-square=9.88; p=0.002). Bone fractures occurred in 5 (0.5%) and 9 (0.9%) pts in S and L arm, respectively ( p=0.29, Fisher exact test). Conclusions: After 2-3 years of adjuvant tam, extended treatment with 5 years of letrozole resulted in significant improvement in DFS compared to the standard duration of 2-3 years of letrozole. Clinical trial information: NCT01064635.

515 Background: The ETNA study showed that substituting P with nab-P did not significantly increase the overall rate of pathological complete response (pCR) (P 18.6%, nab-P 22.5%, p = 0.19). The multivariate analysis revealed that tumor subtype (triple negative vs luminal B-like) was the most significant factor (OR 4.85) influencing treatment outcome (Gianni L et al, JAMA Oncol 2018). Methods: This multicenter open label study (NCT01822314) in collaboration with GEICAM and BCRC-WA randomized 695 patients with centrally-confirmed HER2-negative breast cancer to nab-P 125 mg/m2 (346 patients) or P 90 mg/m2 (349 patients). The two drugs were given on weeks 1, 2 and 3 followed by 1-week rest for 4 cycles before 4 cycles of an anthracycline regimen as per investigator choice. The primary endpoint was pCR (absence of invasive cells in breast and nodes). A secondary endpoint is event-free survival (EFS) defined as the time from randomization to the first date of disease progression while on primary therapy or disease recurrence (local, regional, distant, invasive contralateral breast cancer) after surgery or death due to any cause. Results: The ITT analysis of the secondary endpoint EFS at 5 years is reported below: Clinical trial information: NCT01822314. Overall 5-year survival was 84.8% after P and 87.3% for nab-P. No serious adverse events were documented during the follow-up. Conclusions: The improved 5-year EFS after nab-P failed to reach statistical significance (unadjusted P = 0.245). In the analysis by subgroup the numerical improvement was almost exclusively observed in luminal B and not in TN tumors. So far the data do not support substitution of P with nab-P in the schedule and doses adopted in the ETNA trial. Additional analyses will be based on ongoing molecular studies.[Table: see text]

544 Background: We investigated the prognostic role of the PAM50 HER2-enriched (HER2-E) subtype in HER2+ early breast cancer enrolled in the randomized Phase III ShortHER trial. Methods: The ShortHER study randomized 1254 HER2+ early breast cancer patients to receive 9 weeks vs 1 year of adjuvant trastuzumab combined with chemotherapy. Gene expression measured using nCounter platform was available for 438 surgical samples. Intrinsic subtyping was determined using the research-based PAM50 predictor. Metastasis-free survival (MFS) was calculated from randomization to distant disease recurrence or death (median follow up 72 months). Uni- and multi-variable analysis were performed using Cox models. Results: PAM50 subtype distribution was: HER2-E 53% (N = 233), Luminal A 20% (N = 87), Luminal B 10% (N = 43), Normal-like 11% (N = 48) and Basal-like 6% (N = 27). HER2-E subtype was associated with hormone receptor-negative status (p < 0.001) and TILs ≥20% (p < 0.001), but not with stage and age ( < or ≥60 yrs). HER2-E subtype was associated with worse MFS vs other PAM50 subtypes overall (HR 2.78, p = 0.001), in the short (HR 2.24, p = 0.046), and in the long arm (HR 4.04, p = 0.011). Multivariable Cox model confirmed the independent prognostic value of HER2-E subtype (Table). HER2-E subtype added significant prognostic value on top of clinicopathological variables (Likelihood ratio test p < 0.001). Conclusions: HER2-E intrinsic subtype is an independent prognostic factor for HER2+ early breast cancer patients treated with adjuvant chemotherapy and trastuzumab. Integration of PAM50 subtype in prognostic algorithms can help refine risk stratification. These findings warrant independent validation. Clinical trial information: NCT00629278. [Table: see text]

Introduction: Management of triple negative breast cancer (TNBC) is still a major challenge: a significant proportion patients relapse despite adjuvant chemotherapy, and the median survival from relapse rarely exceeds 1 year. Angiogenesis activation as well as impairment in mechanisms of DNA repair are frequently described in sporadic TNBCs, but the role of angiogenesis inhibitors and platinum salts is still controversial. This is a phase II trial of preoperative carboplatin-paclitaxel in combination with bevacizumab in TNBC patients with previously untreated, stage II-III disease. The primary aim of the study is the pathologic complete response (pCR) rate. Among the secondary aims: safety, breast conserving surgery rate, early response assessment with dynamic contrast-enhanced Magnetic Resonance Imaging (DCE-MRI), biomarker analyses. Methods: Patients with hormone receptor negative (ER and PgR Results: the accrual of the 44 planned patients is completed. Patients characteristics were as follows: median age 46 yrs (29-74); clinical stage IIA: 38.2%, IIB: 35.3%, III: 26.5%; ductal histology: 89%; histologic grade 3: 95%. Notably, 59% of the patients had clinically involved axillary nodes. At present, 33 patients are assessable for toxicity. Bevacizumab-associated adverse events (AEs) were mild: grade 1-2 hypertension occurred in 3 patients (9%) and grade 1 bleeding in 9 patients (27%). No grade 4 non-hematologic AEs were recorded; Grade 3 AEs were: liver function tests abnormalities in 3 patients, diarrhea in 3 patients, and neuropathy in 1 patient. Thirty-one patients underwent surgery, 16 patients (51%) received breast conserving surgery. A pCR in breast and axillary lymph-nodes was achieved in 16 patients (51%); 25 patients (81%) had negative axillary nodes (yN0). Conclusions: No unexpected toxicity was observed by combining bevacizumab to neoadjuvant platinum-taxane based CT. This combination is active in TNBCs, and the rate of pCR is in the expected range. Moreover, 80% of the patients have nodal negativity at surgery, and these results are of particular interest taking into account the high proportion of patients with clinically involved nodes at the time of diagnosis. The final results along with biomarkers data and early response assessment with DCE-MRI will be available at the time of the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-14-03.

Purpose Trastuzumab and chemotherapy is the current standard of care in HER2+ early or locally advanced breast cancer, but there are scanty literature data of its real world effectiveness. Methods We retrospectively reviewed 205 patients with HER2+ breast cancer diagnosed in 10 Italian Medical Oncology Units between July 2003 and October 2011. All patients received neoadjuvant systemic therapy (NST) with trastuzumab in association with chemotherapy. Many different chemotherapy regimens were used, even if 90 % of patients received schemes including anthracyclines and 99 % received taxanes. NST was administered for more than 21 weeks (median: 24) in 130/205 (63.4 %) patients, while trastuzumab was given for more than 12 weeks (median: 12 weeks) in 101/205 (49.3 %) patients. pCR/0 was defined as ypT0+ypN0, and pCR/is as ypT0/is+ypN0. Results pCR/0 was obtained in 24.8 % and pCR/is in 46.8 % of the patients. At multivariate logistic regression, nonluminal/HER2+ tumors (P

To gain consensus on the role of bevacizumab plus paclitaxel as first-line treatment for HER2-negative metastatic breast cancer, a panel of expert oncologists experienced in treating patients with metastatic breast cancer in Italy participated in a Delphi consensus study. The panel reached a full consensus on the efficacy of bevacizumab plus paclitaxel and the clinical meaningfulness of the progression-free survival benefit compared with paclitaxel alone, despite the lack of an overall survival effect in clinical trials. The participants agreed that real-world data support the effectiveness and well-defined safety profile of the regimen. Views on the use of bevacizumab plus paclitaxel in specific patient populations were not unanimous and clinical judgment remains important. Nevertheless, a high level of agreement was reached.

Estrogen synthesis suppression induced by aromatase inhibitors in breast cancer (BC) patients may be affected by single nucleotide polymorphisms (SNPs) of the gene encoding aromatase enzyme, CYP19A1. We assessed the association between plasma estrone sulfate (ES), letrozole treatment, and four SNPs of CYP19A1 gene (rs10046 C>T, rs4646 G>T, rs749292 C>T, rs727479 T>G) which seem to be related to circulating estrogen levels. Patients were enrolled into a prospective, Italian multi-center clinical trial (Gruppo Italiano Mammella, GIM-5) testing the association of CYP19A1 SNPs with the efficacy of letrozole adjuvant therapy, in postmenopausal early BC patients. SNPs were identified from peripheral blood cell DNA. Plasma ES concentrations were evaluated by Radio Immuno Assay. Blood samples were obtained immediately before letrozole therapy (N = 204), at 6-weeks (N = 178), 6 (N = 152) and 12-months (N = 136) during treatment. Medians (IQR) of ES were 160 pg/mL (85–274) at baseline, 35 pg/mL (12–64) at 6-weeks, 29 pg/mL (17–48) at 6 months and 25 pg/mL (8–46) after 12 months treatment. No statistically significant association was evident between polymorphisms and ES circulating levels during letrozole therapy. Letrozole suppression of the aromatase enzyme function is not affected by polymorphisms of CYP19A1 gene in postmenopausal BC patients.

Introduction: Several large randomized trials have shown the superiority of combining trastuzumab with chemotherapy versus chemotherapy alone as adjuvant treatment for HER2+ breast cancer patients. We are running a large phase III trial comparing two different trastuzumab durations (Short-HER study). We are reporting the preliminary cardiac safety data. Methods: The Short-HER study is a phase III, multicentric, Italian trial where 2500 HER2+ breast cancer patients will be randomized to: Arm A (Long) 4 courses of anthracycline based chemotherapy (AC or EC) followed by 4 courses of docetaxel in combination with trastuzumab, followed by 14 additional courses of 3-weekly trastuzumab; or Arm B (Short) 3 courses of 3-weekly docetaxel in combination with weekly trastuzumab followed by FEC x3. this is a non-inferiority trial with DFS and OS as primary end points, and 2-yr failure rate and incidence of cardiac events as secondary end points. Left ventricular ejection fraction (LVEF) is measured at baseline, at the end of each sequence of chemotherapy in both arms, and after 9 and 12 months since randomization thereafter. A cardiac event (CE) was defined as the occurrence of any of the followings: 1) LVEF decrease of more than 15 percentage points from baseline ; 2) LVEF decrease of more than 10 percentage points with absolute value below 50%; 3) symptomatic cardiac failure; 4) other cardiac side effects of grade 2 or more. Results: 510 patients from 69 Italian centers have been randomized so far, 251 in arm A (long) and 259 in Arm B (Short). 146 patients enrolled in arm A and 150 patients enrolled in arm B have received at least 3 months of therapy, and are eligible for the present analysis. In arm A (long), 20 patients (13.7%) experienced a CE: 12 patients experienced a LVEF decline of > 15 percentage points (5 patients with LVEF below 50%, one patient with concomitant atrial fibrillation); 3 patients had a LVEF decline of >10 percentage points with an absolute value below 50%. One patient had symptomatic cardiac failure. Two patients developed Grade 2 hypertension. Two patients developed Grade 2 arrhythmia. In arm B (short), 11 patients (7.3%) experienced a CE: 7 patients had a LVEF decline of > 15 percentage points; one patient had a LVEF decline of > 10 percentage points with an absolute value below 50%. Three patients developed Grade 2 arrhythmia Conclusions: This is a non-inferiority study designed on the assumption that a shorter treatment duration is associated with a significantly lower incidence of cardiac events. With 9 clinically relevant CEs (symptomatic cardiac failure or LVEF below 50%) in arm A (Long) and 1 in arm B (Short), these preliminary data support the assumption, and recruitment is ongoing. Supported by Agenzia Italiana del FArmaco (AIFA). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-12-05.

Background: Docetaxel (T; Taxotere) with capecitabine (X) is active against metastatic breast cancer (MBC); bevacizumab (B) has demonstrated efficacy with taxanes in the first-line setting. This study was conducted to assess the efficacy and safety of TX-B in patients (pts) with MBC. Patients and methods: In this single-arm, multicenter phase II study, pts received first-line bevacizumab 15 mg/kg and docetaxel 60 mg/m2 on day 1, plus capecitabine 900 mg/m2 twice per day on days 1-14 every 21 days. The treatment was administrated for 3 cycles and in case of objective response or stable disease at that time, pts were treated with 3 additional cycles. More courses of chemotherapy were administered at Investigator's discretion. Bevacizumab was continued until progressive disease, patient refusal, or unacceptable toxicity. Primary end point was progression-free survival (PFS) and secondary end points were tumor response rate (RR), overall survival (OS), and toxicity. Results: We report data from the first 30 pts enrolled. Median age was 54 (37-72). Eight (27%) pts had triple-negative disease, while 18 (60%) were hormone-receptor positive. HER2 status was recognized as negative in 25 (84%) pts. TX-B was administered for a median of seven cycles. Five complete and 13 partial responses were observed (overall RR 60%). Median response duration was 12 months. Median OS and PFS were 26 and 11 months, respectively. Grade 3/4 adverse events included tromboembolism (10%), neutropenia (23%), hand-foot syndrome (13%), stomatitis (10%). The median TX doses administered per cycle were 60 mg/m2 and 660 mg/m2, respectively. Ten (33%) pts required dose reductions of docetaxel, while capecitabine dose was reduced in 15 (50%) pts. Results of all the 80 assessable pts entered in the study will be presented at the meeting. Conclusion: TX-B demonstrated significant activity with an acceptable toxicity profile. Maintenance therapy with B is possible for a long period of stable tumor disease. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-17-01.

507Background: Neoadjuvant trials with chemotherapy plus anti-HER2 agents consistently showed a lower rate of pathologic complete response (pCR) in HER2+/HR+ vs HER2+/HR-tumors. Here we report the ...