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Background: Palbociclib might be less effective than ribociclib in HR+/HER2- and HER2-enriched BC. This hypothesis is currently being tested in the HARMONIA prospective phase III clinical trial (NCT05207709). Here, we evaluated the downstream biological effects of both drugs using cell lines and patient tumor samples. Methods: Three HR+ BC cell lines (i.e., MCF7, T47D and BT474) were treated at 2 different dose levels of palbociclib or ribociclib (i.e., 100 nM and 500 nM) +/- fulvestrant (1 nM) for 72 hours (h). PAM50 gene signatures were determined on the nCounter, as well as phosphorylation of RB (p-RB) by Western Blot and senescence-associated β-galactosidase activity by FACS. In vitro experiments were performed in triplicates. PAM50 gene signatures were obtained from 49 paired baseline versus week-2 samples and 49 paired baseline versus surgery samples of the CORALLEEN phase II study (Prat, Lancet Oncol. 2020), which treated 49 women with PAM50 Luminal B HR+/HER2- early BC with neoadjuvant ribociclib (600 mg daily) plus endocrine therapy (ET). PAM50 signature scores were also evaluated in publicly available data from 23 paired baseline versus week-2 samples and 16 paired baseline versus surgery samples of the NEOPALANA phase II trial (Ma, Clin Cancer Res. 2017) which treated 50 patients with HR+/HER2- early BC with palbociclib (125 mg daily) plus anastrozole. Changes in PAM50 signatures upon CDK4/6i were determined by paired t-tests and significant analysis of microarray (SAM). Results: Across all cell lines, both palbociclib and ribociclib statistically significantly reduced p-RB at 72h with both doses (i.e., 100 and 500 nM) compared to non-treated cells (p< 0.001). Senescence was also observed at 72h with both doses. Both drugs +/- ET significantly increased the Luminal A signature and decreased Luminal B and proliferation signatures with both doses. However, the HER2-enriched signature was only significantly reduced when both CDK4/6 inhibitors were given at 500 nM. In tumor samples from the CORALLEEN and NEOPALANA phase II studies, a similar change in PAM50 biology was observed with both drugs, namely an increase in Luminal A signature and a decrease in Luminal B and proliferation signatures after 2 weeks of treatment and at surgery. At 2 weeks of treatment, the HER2-enriched signature was significantly decreased in both studies with ribociclib (p< 0.001) and palbociclib (p< 0.001). However, the decrease in the HER2-enriched signature was only observed in surgical samples of patients treated with ribociclib (p< 0.001), but not palbociclib (p=0.194). A difference in sample size could explain this result. Nevertheless, in CORALLEEN, the median number of days between the last dose of ribociclib and surgery was 13.1 days (range: 1-78). In NEOPALANA, the median number of days between the last dose of palbociclib and surgery was 29 days (range: 8-49), except for 8 patients who received additional 10-12 days of palbociclib immediately before surgery (Ma, Clin Cancer Res. 2017). In patients who underwent surgery at 8 days or before, the HER2-enriched signature was significantly decreased for both ribociclib (p< 0.001) and palbociclib (p=0.013). Interestingly, in patients that underwent surgery after >8 days from the last dose, a significant reduction of the HER2-enriched signature was only observed with ribociclib (p< 0.001), but not with palbociclib (p=0.500). Conclusions: Both palbociclib and ribociclib have similar effects on PAM50 biology when given at the same dose. However, in clinical practice, palbociclib is given at a lower dose than ribociclib, and although HER2-enriched signature is significantly decreased in tumors after 2 weeks of CDK4/6i+ET, this effect is only maintained at later time points with ribociclib, indicating a dose-dependent efficacy of CDK4/6i in this biologically aggressive subtype. Citation Format: Natàlia Lorman-Carbó, Olga Martínez-Sáez, Aranzazu Fernandez-Martinez, Patricia Galván, Nuria Chic, Barbara Adamo, Maria Vidal, Montserrat Muñoz, Charles M. Perou, Joaquín Gavilá, Tomás Pascual, Aleix Prat, Fara Brasó-Maristany. Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) [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 P1-13-16.

Supplementary Data

Purpose:We do not yet have validated biomarkers to predict response and outcome within hormone receptor–positive/HER2-positive (HR+/HER2+) breast cancer. The PAM50-based chemo-endocrine score (CES) predicts chemo-endocrine sensitivity in hormone receptor–positive/HER2-negative (HR+/HER2−) breast cancer. Here, we evaluate the relationship of CES with response and survival in HR+/HER2+ breast cancer.Experimental Design:Intrinsic subtype and clinicopathologic data were obtained from seven studies in which patients were treated with HER2-targeted therapy either with endocrine therapy (ET) or with chemotherapy (CTX). CES was evaluated as a continuous variable and categorically from low to high scores [CES-C (chemo-sensitive), CES-U (uncertain), and CES-E (endocrine-sensitive)]. We first analyzed each dataset individually, and then all combined. Multivariable analyses were used to test CES association with pathologic complete response (pCR) and disease-free survival (DFS).Results:A total of 457 patients were included (112 with ET and 345 with CTX). In the combined cohort, CES-C, CES-U, and CES-E were identified in 60%, 23%, and 17% of the patients, respectively. High CES (i.e., CES-E) was associated with a lower probability of achieving pCR independently of clinical characteristics, therapy, intrinsic subtype, and study (adjusted OR = 0.42; P = 0.016). A total of 295 patients were analyzed for DFS with a median follow-up of 66 months. High CES was also associated with better DFS (adjusted HR, 0.174; P = 0.003) independently of pCR, clinical characteristics and intrinsic subtype. In patients with residual disease, the adjusted DFS HR of CES was 0.160 (P = 0.012).Conclusions:In HER2+/HR+ breast cancer, CES is useful for predicting chemo-endocrine sensitivity and provides additional prognostication beyond intrinsic subtype and clinicopathologic characteristics.

PURPOSE CALGB 40603 ( NCT00861705 ), a 2 × 2 randomized phase II trial, demonstrated that adding carboplatin or bevacizumab to weekly paclitaxel (wP) followed by doxorubicin and cyclophosphamide significantly increased the pathologic complete response (pCR) rate in stage II-III triple-negative breast cancer. We now report long-term outcomes (LTOs) and correlative science end points. PATIENTS AND METHODS The Kaplan-Meier method was used to estimate LTOs in 443 patients who initiated study treatment. Log-rank tests and Cox proportional hazards models evaluated the impact of clinical characteristics, pathologic response, calculated residual cancer burden (RCB) in patients with residual disease (RD), treatment assignment, and dose delivery during wP on LTOs, including event-free survival (EFS). Genomic predictors of treatment response and outcomes were assessed on pretreatment tumor samples by mRNA sequencing. RESULTS Among baseline characteristics, only the clinical stage was associated with LTOs. At a median follow-up of 7.9 years, LTOs were not significantly improved with either carboplatin or bevacizumab, overall or in patients with basal-like subtype cancers by genomic analysis. Patients with pCR (n = 205, 46.3%) had significantly higher 5-year EFS (85.5% v 56.6%, log-rank P < .0001) and overall survival (87.9% v 63.4%, P < .0001) rates compared with patients with RD, even those with RCB class I. Among clinical and genomic features, evidence of immune activation, including tumor-infiltrating lymphocytes and low B-cell receptor evenness, was associated with pCR and improved EFS. CONCLUSION Despite higher pCR rates, neither carboplatin nor bevacizumab appeared to improve LTOs although the study was not powered to assess these secondary end points. pCR was associated with superior LTOs even when compared with minimal RD. Markers of immune activation in pretreatment tumor biopsies were independently associated with higher pCR rates and improved survival.

Background PIK3CA mutations have been described in 20-25% of early-stage HER2-positive breast tumors [1], and are associated with reduced pathologic complete response (pCR) rate after chemotherapy and anti-HER2 agents [2]. However, the independence of this finding and association with long-term outcomes within HER2+ patients is still largely unknown. Here, we studied the prognostic implications of PIK3CA mutations by hormone receptor (HR) status and intrinsic subtype in patients with early stage HER2+ breast cancer enrolled in CALGB 40601. Method In CALGB 40601, gene expression profiling by RNA sequencing (RNAseq) with PAM50-determined intrinsic subtype and PIK3CA mutations from whole exome sequencing (WES) were obtained from 184/305 (60%) pretreatment core biopsies. We examined the association of PIK3CA mutations with pCR and event free survival (EFS) by HR status and intrinsic subtype using logistic and Cox regression analyses. Results PIK3CA mutations were found in 32 patients (32/184, 17%). The most frequent mutation was H1047R (12/32,38%), followed by E545K (7/32,22%) and E542K (5/32,16%). PIK3CA mutations were present in 20% and 15% of HR-positive and HR-negative BC subpopulations, respectively. Within Luminal-B, Luminal-A and HER2-Enriched breast tumors, PIK3CA mutations occurred in 36%, 10% and 19% respectively. In the overall population there was lower rate of pCR in mutated-PIK3CA patients than wild-type (34% vs 49%). Using only the subset of patients treated with neoadjuvant trastuzumab-based therapy as standard of care (excluding the lapatinib plus paclitaxel arm), we found a statistically significant lower pCR rate among PIK3CA-mutated tumors using logistic regression model (30% vs 54%, OR=0.30, p=0.045). At a median follow-up of 9.1 years, the presence of PIK3CA mutation was significantly associated with worse EFS in the overall study population (HR 2.58, 95% CI 1.24- 5.35, p=0.011). In a multivariable model including pCR status, HR status and intrinsic subtype (HER2-E vs. not), PIK3CA mutation was independently and significantly associated with worse EFS (HR 2.18, 95% CI 1.04- 4.56, p=0.039). The negative impact of PIK3CA mutation on EFS was statistically significant only in patients with HR-positive (HR 3.6, 95% CI 1.45-8.96, p=0.06) and luminal breast tumors (HR 4.84, 95% CI 1.08-21.7, p=0.039), but not in HR-negative and non-luminal subtypes. Conclusion In our study, the presence of PIK3CA mutation was significantly associated with lower pCR rates in patients treated with chemotherapy plus trastuzumab. Moreover, in uni- and multivariable Cox models, PIK3CA mutations were associated with worse long-term survival, which appeared to be driven by HR-positive and luminal HER2-positive breast tumors. References 1. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumors. Nature 2012;490:61–70. 2. Loibl S, Majewski I, Guarneri V, Nekljudova V, Holmes E, Bria E, et al PIK3CA mutations are associated with reduced pathological complete response rates in primary HER2-positive breast cancer: pooled analysis of 967 patients from five prospective trials investigating lapatinib and trastuzumab. Ann Oncol 2016;27:1519–25. Citation Format: Paola Zagami, Aranzazu Fernandez-Martinez, Naim U. Rashid, Katherine A Hoadley, Patty Spears, Charles M. Perou, Lisa Carey. Prognostic implications of PIK3CA mutation by hormone receptor status and intrinsic subtype in early stage HER2-positive breast cancer: a correlative analysis from CALGB 40601. [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 PD18-04.

Background Hormone receptor–positive/HER2-negative (HR+/HER2-) breast cancer (BC) is associated with low % of stromal tumor-infiltrating lymphocytes (sTIL) and immune gene expression and poor response to immune checkpoint inhibitors. Evaluating the effect of letrozole and ribociclib (L+R) on the immune microenvironment may suggest new opportunities for immunotherapy-based approaches for HR+/HER2- BCs. Here, we present an exploratory correlative analysis from CORALLEEN, a trial that evaluated the efficacy of L+R (vs. chemotherapy [CHT]) in patients with high-risk PAM50 Luminal B BC (Prat et al. Lancet Oncology. 2022). Methods CORALLEEN is a randomized exploratory study in postmenopausal women with operable stage I-III breast cancer, HR+/HER2- and Luminal B by Prosigna®. Patients were randomized 1:1 to receive either 6 cycles of ribociclib (600mg; 3-weeks-on/1-week-off) plus daily letrozole or CHT: 4 cycles of AC followed by 12 doses of weekly paclitaxel. The primary endpoint was the rate of PAM50 Risk of Relapse (ROR)-low score at surgery in each arm. Samples were prospectively collected at baseline, day 15, and surgery. sTILs score, ki67 IHC and gene expression analysis were determined in all available samples. Complete cell cycle arrest (CCCA) was defined as Ki67≤2.7%. Gene expression profiling by mRNA sequencing (RNAseq) was evaluated. We applied a collection of 194 immune- gene expression signatures (iGES), representing multiple biological pathways and cell types, including. Results 106 patients were randomly assigned to receive neoadjuvant L+R (n=52) or CTH (n=54). Overall, Ki67, sTILs and RNA-seq was available in 95.4%, 96.7% and 83.1% of the samples across the 3 time-points. In terms of cell-cycle inhibition, L+R achieved a significant decrease in Ki67 protein expression and led to higher rates of CCCA at 2 weeks (89.6% vs. 43.2%, p< 0.001) and surgery (45.9% vs 25.5%, P=0.054) compared with CHT. Interestingly, the 11-gene PAM50 proliferative score was significantly lower in tumors with CCAA than in those with non-CCCA (p< 0.001) after L+R, but not after CTH (p = 0.682). In contrast, tumors with CCCA after CHT had a significantly lower rate of tumor cellularity compared to tumors with non-CCAA (p = 0.002). This was not observed in the L+R arm (p=0.141). Compared to baseline, no clear and significant patterns in % of sTILs were observed at week 2 and surgery. However, % of TILs at surgery in tumors with CCCA after CHT was higher than in tumors with non-CCCA (median 15% versus 1%, p=0.017). This was not observed in the L+R arm (median 1% and 5%, p=0.584). Interestingly, this inverse relationship between immune infiltration and CCAA was further confirmed by RNA- CHT compared to tumors with non-CCCA, whereas 174 (89.7%) of iGES were upregulated (FDR< 5%) in tumors with non-CCCA after L+R compared to tumors with CCCA. Finally, L+R and CTH treatment at week 2 and surgery showed an increase in adaptive immune signatures indicative of activated T-cell and B-cell phenotypes; however, CTH was uniquely associated with increased cytokine signaling, enhanced antigen presentation, dendritic, granulocyte, macrophage and NK cells and decrease in Th17, Th2 and Treg cells. Conclusion In early-stage Luminal B breast cancer, L+R induce a potent anti-proliferative effect compared to CHT. Both treatments generally increased T- and B-cell immune infiltration; however, an inverse relationship between immune infiltration and anti-proliferative response at surgery exists according to treatment, where immune infiltration is increased in residual tumors with non-CCAA when treated with L+R, but the opposite is observed with CHT. The prognostic value of immune and anti-proliferative effects of L+R in residual tumors is currently being evaluated in the prospective RIBOLARIS phase II clinical trial (NCT05296746). Citation Format: Tomás Pascual, Nuria Chic, Aranzazu Fernandez-Martinez, Blanca González-Farré, Laia Paré, Cristina Saura, Cristina Hernando, Montserrat Muñoz, Míriam Arumí, Patricia Galván, Xavier Gonzalez-Farré, Mafalda Oliveira, Miguel Gil Gil, Eva Ciruelos, Patricia Villagrasa, Joaquin Gavila Gregori, Aleix Prat, Charles M. Perou. Cell-Cycle Inhibition and Immune Microenvironment in HR+/HER2- Breast Cancer During and After preoperative ribociclib and letrozole vesus chemotherapy: A correlative analysis of the 1402-SOLTI/CORALLEEN phase 2 trial [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 PD17-07.

PURPOSE CALGB 40601 assessed whether dual versus single human epidermal growth factor receptor 2 (HER2) –targeting drugs added to neoadjuvant chemotherapy increased pathologic complete response (pCR). Here, we report relapse-free survival (RFS), overall survival (OS), and gene expression signatures that predict pCR and survival. PATIENTS AND METHODS Three hundred five women with untreated stage II and III HER2-positive breast cancer were randomly assigned to receive weekly paclitaxel combined with trastuzumab plus lapatinib (THL), trastuzumab (TH), or lapatinib (TL). The primary end point was pCR, and secondary end points included RFS, OS, and gene expression analyses. mRNA sequencing was performed on 264 pretreatment samples. RESULTS One hundred eighteen patients were randomly allocated to THL, 120 to TH, and 67 to TL. At more than 7 years of follow-up, THL had significantly better RFS and OS than did TH (RFS hazard ratio, 0.32; 95% CI, 0.14 to 0.71; P = .005; OS hazard ratio, 0.34; 95% CI, 0.12 to 0.94; P = .037), with no difference between TH and TL. Of 688 previously described gene expression signatures, significant associations were found in 215 with pCR, 45 with RFS, and only 22 with both pCR and RFS (3.2%). Specifically, eight immune signatures were significantly correlated with a higher pCR rate and better RFS. Among patients with residual disease, the immunoglobulin G signature was an independent, good prognostic factor, whereas the HER2-enriched signature, which was associated with a higher pCR rate, showed a significantly shorter RFS. CONCLUSION In CALGB 40601, dual HER2-targeting resulted in significant RFS and OS benefits. Integration of intrinsic subtype and immune signatures allowed for the prediction of pCR and RFS, both overall and within the residual disease group. These approaches may provide means for rational escalation and de-escalation treatment strategies in HER2-positive breast cancer.

Background: Patients with HER2+ breast cancer who have residual disease after neoadjuvant anti-HER2 plus chemotherapy have a high risk of recurrence and benefit from adjuvant trastuzumab emtansine (T-DM1). We hypothesize that endocrine-responsive residual tumors after neoadjuvant treatments may have good outcomes among patients receiving only adjuvant endocrine therapy plus trastuzumab. Using paired pre- and post-treatment samples from CALGB 40601 and other neoadjuvant cohorts that did not include adjuvant T-DM1, we investigated survival by pretreatment and residual disease ESR1 and PgR gene expression in CALGB 40601, and by estrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry (IHC) in the other cohorts. We considered endocrine-responsive tumors those with ER and/or PR expression by gene expression or IHC. Methods: CALGB 40601 is a randomized neoadjuvant trial of single vs. dual HER2-targeting (trastuzumab and/or lapatinib added to paclitaxel). We only included those patients who had not suffered from disease progression or death during their preoperative treatments. In total, 77 patients with paired pretreatment and residual disease tumors were profiled by mRNA sequencing and studied. Cutoffs for ESR1 and PgR mRNA expression mimicking clinical positivity were obtained using 265 pretreatment CALGB 40601 tumors (and 1,045 TCGA samples for ESR1). We also examined ER and PR IHC in paired tumors from 202 patients treated at 4 different collaborating institutions; all had residual disease after neoadjuvant HER2 targeting plus chemotherapy. We considered ER- or PR-positive as ≥10% positively staining cells. The primary endpoint was EFS, defined as the time from randomization to event in CALGB 40601 and from the first systemic therapy to event in the 4-institution validation cohort. Results: In 77 patients from CALGB 40601 with paired (pretreatment/residual disease) specimens, 38 (49.3%) had ESR1+/ESR1+ tumors. The EFS was superior in the ESR1+/ESR1+ (n=38) group than in the remaining others (the log-rank test, p=0.011). The 5- and 7-year EFS rates for the ESR1+/ESR1+ (n=38) were 92.1% and 89.2%, whereas the rates were uniformly < 70% in the others. In particular, the 5-year EFS rate among 11 patients with ESR1+/ESR1- tumors was 61.4%. This remained significant in multivariable analysis with clinical stage and treatment arm; the hazard ratio (HR) for EFS in ESR1+/ESR1+ versus all others was 0.29 (95% CI, 0.09-0.90). In ESR1+/ESR1+ tumors, 5-year EFS rates were high for those whose residual disease and also being PgR+ (n=32) or PAM50 LumA or Normal-like (n=34) (93.8% and 97.1%, respectively). In the institutional validation cohort, pretreatment /residual disease ER(+)/ER(+) tumors (n=113) had superior 3-year EFS versus all others (p=0.010). At a median follow-up of 35.9 months, the 3-year EFS rates for ER(+)/ER(+) and all other groups were 96.6% and 82.6%, respectively. This remained strongly significant in multivariable analysis with clinical stage; the HR for EFS in ER(+)/ER(+) versus all other groups was 0.28 (95% CI, 0.09-0.88). Among 46 who also had PR+ in the residual disease, the 3-year EFS was 100.0%. Conclusions: HER2+ patients with ER+ pretreatment and ER+ residual disease after neoadjuvant chemotherapy + HER2-targeting have a very good survival outcome despite not receiving additional anti-HER2 targeting with T-DM1. This may provide a simple mechanism to better tailor therapy within residual disease patients using serial ER measurements. Citation Format: Sung Gwe Ahn, Aranzazu Fernandez-Martinez, Mei-Yin C. Polley, Soong June Bae, Seho Park, Núria Chic, Fara Brasó-Maristany, Benedetta Conte, Valentina Guarneri, Maria Vittoria Dieci, Gaia Griguolo, PierFranco Conte, Joon Jeong, Aleix Prat, Lisa A. Carey, Charles M Perou. A good prognosis of endocrine-dependent tumors among residual invasive cancer after anti-HER2 therapy: CALGB 40601 (Alliance) and validation studies [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 PS10-02.

Introduction: The ALTERNATE trial randomized postmenopausal women with ER Allred 6-8 HER2- breast cancer to 6 months of NET with anastrozole (A), fulvestrant (F) or the combination (A+F). Biopsies were taken preNET and after 4-weeks(wks). Patients with Ki67 values >10% at 4-wks were offered triage to neoadjuvant chemotherapy. Patients with on-treatment Ki67 ≤ 10% who completed NET underwent surgery and Ki67 was reassessed. The primary endpoint was endocrine-sensitive disease rate (ESDR). ESD is defined as pCR or PEPI-0 residual disease (pT1-2, pN0, Ki67 ≤ 2.7%). We previously reported that the ESDR difference between the F-containing arms and the A arm was not >10% (ASCO 2020) and that baseline RNA-seq-based intrinsic subtypes predicted outcomes overall (SABCS 2021). Herein we describe relationships between PAM50 intrinsic subtype and Ki67 values by treatment arm because comparative drug effectiveness in adjuvant endocrine therapy studies in ER+ HER2- breast cancer can be predicted by the degree of Ki67 suppression (PMC3518447). Methods: 743 of the 1297 eligible patients (A: 264; F: 231; A+F: 248) had RNA extracted from preNET frozen tumor biopsies with >50% tumor content and subjected to RNA seq. Intrinsic subtypes were then assigned as LumA, LumB, and NonLum (Basal or HER2-E) using open-source PAM50-based informatics. Differences in the proportion with wk4 Ki67 > 10%, % change in wk4 ki67, and surgical CCCA (Ki67 ≤ 2.7%) rate (sxCCCA) between treatments and by intrinsic subtype was assessed using stratified logistic regression, Wilcoxon rank sum test, and Fisher’s exact test, respectively. Analysis of sxCCCA excluded those who failed to complete NET for reasons other than disease progression or early Ki67 >10%. Results: Amongst the 358 LumA cases there were no significant differences in Ki67-based endpoints between treatments. Among the 292 LumB cases, the wk4 ki67 > 10% rate was lower with A+F (19.4%) than A (43%) (P=0.0002) and was somewhat lower in F (31%) versus A (P=0.076). The % change in wk4 Ki67 in LumB cases, adjusted for baseline Ki67, showed markedly superior suppression for A+F versus A (-90% vs. -77%; P= Conclusion: The combination of A+F was significantly more effective than either drug alone for the control of LumB breast cancer cell proliferation. This suggests that A+F may be a more effective adjuvant endocrine therapy than A alone in LumB disease. The lower Ki67 suppression with A alone also suggests that poorer outcome in some LumB tumors may be due to insufficient ER targeting rather than ER-independent tumor growth Support: U10CA180821, U10CA180882, U24CA196171, UG1CA189856, U10CA180868 (NRG), NCI BIQSFP, BCRF, Genentech, AstraZeneca. https://acknowledgments.alliancefound.org. (MJE) CPRIT RR140033, P50-CA186784, P50-CA58223, U01-CA214125, U24-CA210954, Gift from Ralph and Lisa Eads, McNair Scholarship. ClinicalTrials.gov Identifier: NCT01953588 Citation Format: Matthew J. Ellis, Meenakshi Anurag, Jeremy Hoog, Aranzazu Fernandez-Martinez, Cheng Fan, Richard Gibbs, Souzan Sanati, Kiran Vij, Mark Watson, Travis Dockter, Olwen Hahn, Joseph Guenther, Abigail Caudle, Erica Crouch, Amy Tiersten, Monica Mita, Wajeeha Razaq, Tina J. Hieken, Yang Wang, A. Marilyn Leitch, Gary W. Unzeitig, Eric Winer, Anna Weiss, Kelly Hunt, Ann H. Partridge, Charles M. Perou, Vera Suman, Cynthia X. Ma, Lisa A. Carey. The effect of intrinsic subtype on inhibition of tumor growth by anastrozole vs. fulvestrant vs. the combination: Results from the Alliance neoadjuvant endocrine therapy (NET) ALTERNATE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT026.

509 Background: Several biologic features are implicated in the differences in response and survival to dual (trastuzumab and lapatinib [HL]) vs. single (trastuzumab [H]) HER2-blockade across neoadjuvant trials in early-stage HER2+ breast cancer. We evaluated the association of intrinsic subtypes and gene expression signatures with pathologic complete response (pCR) and event-free survival (EFS) in a pooled analysis of three independent phase III neoadjuvant studies with similar designs: CALGB 40601 (Alliance), NeoALTTO, and NSABP B-41. Methods: Gene expression profiling by RNA sequencing was assessed on 761 pre-treatment samples (264 from CALGB 40601, 249 from NeoALTTO, 248 from NSABP B-41). Intrinsic subtypes and 759 gene expression signatures were calculated. We studied the association of pCR and the benefit of dual (HL) vs. single (H) HER2-blockade by tumor intrinsic subtype in the pooled set. The ability of multiple gene expression signatures to predict pCR and EFS across the three studies was also tested by logistic and Cox regression analyses. Results: pCR status was associated with EFS only in HER2-Enriched (HR 0.45, 95% CI 0.29-0.71, p-value < 0.001) and Basal-like (HR 0.19, 95% CI 0.04-0.86, p-value 0.031) intrinsic subtypes, but not in Luminal and/or ER+ tumors. The EFS benefit of dual vs. single HER2-blockade was limited to HER2-Enriched tumors (HR 0.47, 95% CI 0.27-0.81, p-value 0.007). When evaluating the three clinical trials separately, we found 89/759 (11.7%) gene expression signatures in common for the prediction of pCR across the three clinical trials, including HER2-amplicon and immune activation signatures. Luminal-related signatures were associated with lower pCR rates but better EFS outcomes, especially in patients with residual disease. Stratified Cox regression models by study showed a significant and strong association of NK, B and plasma cells, as well as Ig-related signatures with a better EFS outcome, while vascular, proliferation, and metastasis signatures were associated with poor EFS. Conclusions: In early-stage HER2+ breast cancer, the relationship between pCR and EFS differs by tumor intrinsic subtype, and the benefit of dual vs. single HER2-blockade seems to be limited to HER2-Enriched subtype tumors. Immune signatures were associated with higher pCR rates and better EFS, luminal signatures were associated with lower pCR rates but good EFS outcomes, and vascular/proliferation/metastasis signatures were associated with poor EFS across the three clinical trials. Clinical trial identification: CALGB 40601: NCT00770809. (CALGB is part of the Alliance for Clinical Trials in Oncology). NeoALTTO: NCT00553358 NSABP B-41: NCT00486668

Purpose:We do not yet have validated biomarkers to predict response and outcome within hormone receptor–positive/HER2-positive (HR+/HER2+) breast cancer. The PAM50-based chemo-endocrine score (CES) predicts chemo-endocrine sensitivity in hormone receptor–positive/HER2-negative (HR+/HER2−) breast cancer. Here, we evaluate the relationship of CES with response and survival in HR+/HER2+ breast cancer.Experimental Design:Intrinsic subtype and clinicopathologic data were obtained from seven studies in which patients were treated with HER2-targeted therapy either with endocrine therapy (ET) or with chemotherapy (CTX). CES was evaluated as a continuous variable and categorically from low to high scores [CES-C (chemo-sensitive), CES-U (uncertain), and CES-E (endocrine-sensitive)]. We first analyzed each dataset individually, and then all combined. Multivariable analyses were used to test CES association with pathologic complete response (pCR) and disease-free survival (DFS).Results:A total of 457 patients were included (112 with ET and 345 with CTX). In the combined cohort, CES-C, CES-U, and CES-E were identified in 60%, 23%, and 17% of the patients, respectively. High CES (i.e., CES-E) was associated with a lower probability of achieving pCR independently of clinical characteristics, therapy, intrinsic subtype, and study (adjusted OR = 0.42; P = 0.016). A total of 295 patients were analyzed for DFS with a median follow-up of 66 months. High CES was also associated with better DFS (adjusted HR, 0.174; P = 0.003) independently of pCR, clinical characteristics and intrinsic subtype. In patients with residual disease, the adjusted DFS HR of CES was 0.160 (P = 0.012).Conclusions:In HER2+/HR+ breast cancer, CES is useful for predicting chemo-endocrine sensitivity and provides additional prognostication beyond intrinsic subtype and clinicopathologic characteristics.

Càncer de mama; Genòmica del càncer; Recerca translacional Cáncer de mama; Genómica del cáncer; Investigación traslacional Breast cancer; Cancer genomics; Translational research Novel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression. This work was supported by the grants from the European Union’s Horizon 2020 Research and Innovation Programme under Grant agreement No. 847912 (to A.P.), the Instituto de Salud Carlos III-PI16/00904 (to A.P.), Pas a Pas (to A.P.), Save the Mama (to A.P.), Breast Cancer Now-2018NOVPCC1294 (to A.P.), Fundación Científica Asociación Española Contra el Cáncer-Ayuda Postdoctoral AECC 2017 (to F.B.-M.), Fundación SEOM, Becas FSEOM para Formación en Investigación en Centros de Referencia en el Extranjero 2018 (to T.P.) and PhD4MD - Departament de Salut expedient SLT008/18/00122 (to N.C.).

Background: HER2-positive (HER2+) breast cancer (BC) comprises all the four PAM50 molecular subtypes. Among these, the HER2-Enriched (HER2-E) appear to be associated with higher pathological complete response (pCR) rates following anti-HER2-based regimens. Here, we present a meta-analysis to validate the association of the HER2-E subtype with pCR following anti-HER2-based neoadjuvant treatments with or without chemotherapy (CT). Methods: A systematic literature search was performed in February 2019. The primary objective was to compare the association between HER2-E subtype (versus others) and pCR. Selected secondary objectives were to compare the association between 1) HER2-E subtype and pCR in CT-free studies, 2) HER2-E subtype within hormone receptor (HR)-negative and HR+ disease and 3) HR-negative disease (versus HR+) and pCR in all patients and within HER2-E subtype. A random-effect model was applied. The Higgins��� I2 was used to quantify heterogeneity. Results: Sixteen studies were included, 5 of which tested CT-free regimens. HER2-E subtype was significantly associated with pCR in all patients (odds ratio [OR] = 3.50, p < 0.001, I2 = 33%), in HR+ (OR = 3.61, p < 0.001, I2 = 1%) and HR-negative tumors (OR = 2.28, p = 0.01, I2 = 47%). In CT-free studies, HER2-E subtype was associated with pCR in all patients (OR = 5.52, p < 0.001, I2 = 0%) and in HR + disease (OR = 4.08, p = 0.001, I2 = 0%). HR-negative status was significantly associated with pCR compared to HR + status in all patients (OR = 2.41, p < 0.001, I2 = 30%) and within the HER2-E subtype (OR = 1.76, p < 0.001, I2 = 0%). Conclusions: The HER2-E biomarker identifies patients with a higher likelihood of achieving a pCR following neoadjuvant anti-HER2-based therapy beyond HR status and CT use. Future trial designs to escalate or de-escalate systemic therapy in HER2+ disease should consider this genomic biomarker.

Background: Neoadjuvant ET (NET) offers an opportunity to assess ET sensitivity for ER+ HER2- BC and potentially to tailor therapy. Ki67 >10% on biopsy after 2-4 weeks (wks) of NET identifies patients (pts) with intrinsic ET resistance; while pathologic complete response (pCR) and modified preoperative endocrine prognostic index of 0 (mPEPI 0: pT1-2N0, Ki67 ≤2.7%) at surgery indicates sensitivity to ET. However, on-NET biopsy is not always acceptable or feasible and delays the ET sensitivity determination. PAM50 ROR score and intrinsic subtypes by tumor RNA profiling are prognostic in pts with early stage ER+ HER2- BC, and predict pCR rates to neoadjuvant chemotherapy (NCT) (PMC2667820). We therefore hypothesized that PAM50 analysis on pre-NET biopsies could predict the likelihood of a) a high on-NET Ki67, b) mPEPI-0 or pCR at surgery and, c) pCR for pts triaged to NCT. Methods: The ALTERNATE trial is a phase III study that randomized postmenopausal pts with clinical stage II/III ER+ (Allred score 6-8) HER2- BC to receive neoadjuvant anastrozole, fulvestrant, or both for 6 months before surgery. Research biopsy was required at pre-NET and wk 4, then optional at wk 12. Pts with Ki67 >10% on biopsy at wk 4 or 12 discontinued NET and were offered NCT. PAM50 intrinsic subtype and ROR-P values were generated from mRNA sequencing (RNASeq) analysis on pre-NET biopsies using open-source informatics (PMC7723687) and evaluated for prediction of on-NET Ki67 >10% at wk 4 or 12, pCR or mPEPI-0 post NET, and pCR post NCT. Results: 749 of 1,297 eligible trial pts were included in the analyses, after excluding 548 pts due to insufficient pre-NET tumor for RNASeq (n=511) or PAM50 normal subtype (n=37). Similar to the entire ALTERNATE population, the rate of Ki67 >10% at wk 4 or 12 was 24.4% (95% CI: 21.4-27.7%) and the rate of mPEPI-0/pCR post NET was 19.8% (95% CI: 17.0-22.8%). There were 393 (52.5%) Lum A, 302 (40.3%) Lum B, and 54 (7.2%) non-Lum (9 Basal, 45 HER2-E) BCs. These included 196 (26.2%) ROR-P low, 354 (47.3%) ROR-P medium and 199 (26.6%) ROR-P high BCs. Both the rates of Ki67 >10% at wk 4 or 12 and mPEPI-0/pCR differed significantly with respect to PAM50 subtype or ROR-P category, such that Lum A or ROR-P low BCs were least likely to have a Ki67 >10% at wk 4 or 12 and most likely to achieve mPEPI-0/pCR (Table). 93 of 168 (55.4%) pts triaged to NCT had RNA-seq results, yielding 26 Lum A, 49 Lum B, 4 Basal and 14 HER2-E, with the pCR rates of 0%, 6.1%, 0%, and 21.4%, respectively. There were 10 ROR-P low, 39 medium, and 44 high tumors, with a pCR rate of 0%, 5.1% and 9.1%, respectively. Conclusion: These data indicate that both baseline ROR-P and intrinsic subtype are predictive of early on-NET Ki67 > 10% and mPEPI 0/pCR at surgery after NET. For pts triaged to NCT based on an early on-NET Ki67 >10%, the HER2-E group had the highest pCR rate (20%) and no pCRs were observed in Lum A. These data may be useful for directing neoadjuvant therapy in postmenopausal pts with ER+ HER2- BC. Support: U10CA180821, U10CA180882, U24CA196171, UG1CA189856, U10CA180868 (NRG), NCI BIQSFP, BCRF, Genentech, AstraZeneca. https://acknowledgments.alliancefound.org. (MJE) CPRIT RR140033, P50CA186784, P50-CA58223, U01 CA214125, U24CA210954, Gift from Ralph and Lisa Eads, McNair Scholarship. Trials.gov Identifier: NCT01953588. Table 1.Rates of Ki67 >10% and mPEPI-0/pCR post NET by PAM50 subtype and ROR-P categoryKi67 >10% at wk 4 or 12mPEPI 0/pCR post NETPAM50 SubtypenYes, n (%)PnNo, n (%)PLum A37251 (13.7%) 95% CI: 10.4-17.6% Citation Format: Cynthia X Ma, Meenakshi Anurag, Travis Dockter, Jeremy Hoog, Aranzazu Fernandez-Martinez, Cheng Fan, Richard Gibbs, Souzan Sanati, Kiran Vij, Mark Watson, Olwen Hahn, Joseph Guenther, Abigail Caudle, Erika Crouch, Amy Tiersten, Monica Mita, Wajeeha Razaq, Tina J Hieken, Yang Wang, A. Marilyn Leitch, Gary W Unzeitig, Anna Weiss, Eric P Winer, Kelly Hunt, Ann H Partridge, Lisa A Carey, Charles M Perou, Matthew J Ellis, Vera Suman. Pam50 intrinsic subtype and risk of recurrence score (ROR) for the prediction of endocrine (ET) sensitivity and pathologic response to chemotherapy in postmenopausal women with clinical stage II/III estrogen receptor positive (ER+) and HER2 negative (HER2-) breast cancer (BC) in the alternate trial (Alliance A011106) [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 PD9-03.

Gene expression profiling has had a considerable impact on our understanding of breast cancer biology. During the last decade, 4 intrinsic molecular subtypes of breast cancer (Luminal A, Luminal B, HER2-enriched [HER2-E] and Basal-like) have been identified and intensively studied. In this article, we review and discuss the clinical implications of the 2 non-luminal subtypes (i.e. HER2-E and Basal-like) identified within hormone receptor (HR)-positive disease. After reviewing 32 studies for a total of 13,091 samples, ∼8% and ∼ 15% of early and metastatic HR+/HER2-negative breast cancer, respectively, were found to be non-luminal. Clinically, HR+/HER2-negative/non-luminal subtypes have been associated with estrogen independence, chemo-sensitivity, resistance to CDK4/6 inhibition and poor outcome. Interestingly, EGFR/HER2 tyrosine kinase inhibition might be of value in the HR+/HER2-negative/HER2-E subtype. Finally, the HER2-E subtype within HR+/HER2 + disease represents ∼ 30% and has been associated with anti-HER2 sensitivity, chemo-sensitivity and resistance to CDK4/6 inhibition. In the upcoming years, retrospective and prospective clinical trials evaluating both biomarkers should lead to improvements in patient outcomes.

Aim: While both efficacy and safety of anti-PD-1 agents seem to be independent of previous treatment with anti-CTLA-4, limited data exist of efficacy and toxicity of ipilimumab after progression on anti-PD-1 therapy. This retrospective analysis describes the efficacy and safety of sequential therapy with ipilimumab in patients with metastatic melanoma who progressed on anti-PD-1 antibody. Methods: Nine patients who progressed on anti-PD-1 therapy received four cycles of ipilimumab 3 mg/kg every 3 weeks. Results: Two out of nine patients (2/9; 22.2%) showed a partial response, and seven patients (7/9; 77.8%) experienced disease progression. Median progression-free survival was 3.14 months (95% CI: 2.56–3.71), and the median overall survival since the start of anti-PD-1 therapy was 16.8 months (95% CI: 8.1–25.4). Five (5/9; 55.6%) patients experienced grade 3 immune-related adverse events. No grade 4 or 5 adverse events were reported. Conclusion: In this small retrospective series of cases, the efficacy of ipilimumab post-anti-PD-1 was similar to that described in the previous reports on ipilimumab.

Purpose: CALGB (now part of the Alliance for Clinical Trials in Oncology) 40603 was a randomized Phase II study investigating pathological complete response rates (pCR) in triple-negative breast cancer (TNBC) patients receiving neoadjuvant weekly paclitaxel followed by doxorubicin and cyclophosphamide +/- bevacizumab and/or carboplatin for which 5-year event-free survival (EFS) results are now available. This is a valuable resource to characterize clinical and genomic features associated with response and survival. Methods: Clinical parameters and pre-treatment tumor biopsy RNA-sequencing (RNAseq) from 295 TNBC patients were analyzed to identify features associated with pCR and/or EFS. A panel of 689 previously published gene expression signatures were evaluated to provide insights regarding potential associations between clinical endpoints and genomically determined cell types and signaling pathway activities. Additionally, B cell receptor (BCR) and T cell receptor (TCR) sequences were examined, and repertoire abundance, richness, and diversity measures were calculated to investigate correlations with pCR and EFS status. Univariate Mann-Whitney U-Tests were used for continuous variables, and Fisher’s test was used for categorical features, with unadjusted p-values < 0.05 designated as significant. Results: While the addition of bevacizumab and carboplatin each significantly improved pCR rates, neither improved EFS. We examined outcomes according to race and no differences were seen for either pCR rate or EFS. 131 features, including high proliferation and multiple interferon signatures were significantly associated with pCR, but not with EFS. Alternatively, 69 features, including clinical factors for T stage and node status, were prognostic for EFS, but not significantly associated with pCR. Nevertheless, pCR itself was the strongest predictor of EFS, and was the only feature significantly associated with EFS after adjusting for multiple comparisons (Benjamini-Hochberg False Discovery Rate = 6.7e-3). In total, 52 genomic features were significantly correlated with both pCR and EFS, 44 of which were features of the immune microenvironment. Immune associated features included signatures of T cells, B cells and NK cells, immune checkpoint pathways (PD-1, PD-L1, CTLA4), and antigen presentation (dendritic cells, MHC-I, MHC-II). In particular, low BCR evenness, which is a measure of uniformity of unique BCR sequence abundance, was strongly associated with both pCR and EFS, suggesting that an antigen-specific adaptive immune response with clonally selected B cells is occurring in patients that have improved response and survival. Furthermore, a multivariate Cox Proportional Hazards model assessing BCR evenness along with age, T stage, N stage, grade and pCR found BCR evenness to be an independent prognostic feature for EFS in TNBC. Conclusions: Evidence of distinct predictors of pCR and EFS in TNBC patients treated with neoadjuvant chemotherapy suggests that, while pCR is still the strongest prognostic feature, high expression of many immune related gene expression signatures in pretreatment tumor samples are promising biomarkers of improved EFS. In addition to the important role of T cells in an anti-tumor response, these data show high IgG expression and evidence of B cell clonal selection associates with improved response and survival, supporting an important role for B cells in the adaptive response that portends a long-term benefit of chemotherapy in TNBC. Support: U10CA180821, U10CA180882, U24CA196171, P50-CA58223, Genentech, and The Breast Cancer Research Foundation. https://acknowledgments.alliancefound.org; ClinicalTrials.gov Identifier: NCT00861705 Citation Format: Jonathan H Shepherd, Terry Hyslop, Cheng Fan, Aranzazu Fernandez Martinez, Joel Parker, Katherine Hoadley, Zhiyuan Hu, Yun Li, Matthew Soloway, Patricia Spears, Ann Partridge, William Sikov, Lisa A Carey, Charles M Perou. Genomic analysis of the CALGB 40603 (Alliance) neoadjuvant trial in TNBC identifies immune features associated with pathological complete response and event-free survival [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 PD9-03.

Immunotherapy (IT) agents and BRAF inhibitors (BRAFi) are effective treatments for patients with advanced BRAF-mutant melanoma although the optimal sequence remains to be elucidated. The aim of this study was to compare the outcomes of two different cohorts of patients treated with BRAFi first, then IT or the reverse sequence.This is a retrospective study on two groups of patients: a cohort was treated first with BRAFi followed by immunotherapy (BRAFi-IT) and the other cohort with the reverse sequence (IT-BRAFi). Baseline characteristics and clinical outcomes were compared between the two cohorts.A total of 25 patients were included in the study. Sixteen patients were given BRAFi-IT sequence and nine received IT-BRAFi sequence. No differences were observed in the characteristics of patients prior to each treatment between cohorts. Objective response rate (ORR) achieved by BRAFi were not different among groups. ORR achieved by IT was higher when administered after BRAFi (43.8 vs 0 %). Survival rates at 1-2 years were similar in both cohorts and median overall survival was not different for BRAFi-IT and IT-BRAFi (log rank test p = 0.97).No differences were observed in OS between the two cohorts. These results support the indistinct use of IT or BRAFi as initial treatment in patients with metastatic BRAF-mutant melanoma, although higher rate of response to IT was observed when administered after BRAFi. Prospective randomized clinical trials are needed on this issue.

Phlegmasia cerulea dolens (PCD) is a rare presentation of deep vein thrombosis (DVT) which clinically mimicks arterial ischemia and is caused by extensive thrombotic occlusion of the venous outflow of a major vein of an extremity. DVT is a common and potentially life-threatening complication in patients with lung cancer. We report the case of a patient who developed PCD in an upper limb while receiving anticoagulation treatment with low-molecular-weight heparin (LMWH) for recurrent venous thromboembolism (VTE) events at presentation of EML4-ALK translocated metastasic lung adenocarcinoma. Crizotinib therapy was associated not only with response of metastatic lesions but with a dramatic improvement of cancer-associated hypercoagulability. To our knowledge, this is the first case reporting PCD in an ALK-rearranged advanced NSCLC patient.

571 Background: In HER2-positive breast cancer, new biomarkers of response are needed in order to direct multi-agent anti-HER2 combinations towards patients in whom they are truly needed. CALGB 40601 and PAMELA trials tested neoadjuvant dual HER2 blockade and included gene expression analysis aimed to evaluate different genomic biomarkers of trastuzumab and/or lapatinib benefit. Methods: Gene expression by mRNA sequencing (RNAseq) was performed on 265 and 142 pre-treatment tumors of the CALGB 40601 and the PAMELA clinical trials respectively. Intrinsic subtypes were determined by nCounter PAM50-predictor on the PAMELA samples. A new HER2-positive specific gene-centering method was trained on the PAMELA RNAseq data, and showed a higher concordance with PAM50 predictions obtained from nCounter platform. This method was then applied to CALGB 40601 samples. Results: In the combined cohort, the subtype distribution was 10% Luminal A, 8% Luminal B, 62% HER2-enriched (HER2-E), 10% Basal and 10% Normal-like. The pCR rate was significantly higher in HER2-E vs. not HER2-E subtypes (48.6% vs. 20.7%; P < 0.001). HER2-E subtype correlation, ERBB2 amplicon and B-cell genomic signatures were associated with pCR, while luminal signatures were associated with non-responders. In multivariate analysis HER2-E subtype, ERBB2 mRNA and IgG signature expression were independent predictors of response to paclitaxel + trastuzumab +/-lapatinib (OR = 1.98, OR = 1.51, OR = 1.48, respectively, P

Currently, limited data exist on the safety of pembrolizumab in patients with metastatic melanoma who have developed severe immune-related adverse events following treatment with ipilimumab. We report a 45-year-old male patient with BRAF-mutant metastatic melanoma who discontinued treatment with ipilimumab due to treatment-related grade 3 colitis and was subsequently treated with the anti-programmed cell death 1 protein (PD-1) antibody pembrolizumab. He has been on treatment with pembrolizumab for more than 20 months with no major toxicities and has achieved an objective partial response, which is ongoing.

Immune checkpoint inhibitors, such as ipilimumab (an anti-CTLA4 antibody), have become a commonly used therapy in cancer. To date, safety data of patients with underlying autoimmune disease is limited. We present a case of a patient with rheumatoid arthritis who was diagnosed of a BRAF-mutant metastatic melanoma. The patient was treated with ipilimumab and presented with high-grade colitis requiring immunosuppressors. Despite of the immune-related adverse event, no exacerbation of the rheumatoid arthritis was observed and the patient achieved a complete response. This case report contributes to the scarce literature on the use of immune checkpoint inhibitors in patients with an underlying autoimmune condition.

66 Background: Anti-PD1 antibodies (Abs) Nivolumab (Nivo) and Pembrolizumab (Pembro) have both demonstrated to improve the overall survival (OS) in patients (pts) with advanced or metastatic melanoma (MM) in the first line setting. We previously reported that an increase in absolute eosinophil counts (AEC) of 100/mm3 over baseline at week 3 and an AEC > 400/mm3 at week 12 during anti-PD1 treatment (tmt) might identify pts with MM most likely to experience long-term disease control (1). AEC could serve as an important marker allowing the detection of those patients who may benefit from the therapy with anti-PD1 Abs. Methods: This retrospective observational study included pts with MM who received anti-PD1 tmt in a single institution. The objective was to identify whether an increase over the upper limit of normal AEC (>400/mm3) at any time during the tmt could predict better outcomes. Blood tests were performed before every tmt administration. Progression free survival (PFS) and OS were evaluated. Descriptive statistics were used to analyze patient baseline characteristics. PFS and OS were estimated by the Kaplan-Meier method. Results: From March 2013 to February 2016, 50 pts were treated with anti-PD1 Abs (39 pts with Pembro and 11 pts with Nivo). Median age was 58 years (33-84). 72% had received previous systemic tmt for MM, 64% had stage M1c disease, and 50% had elevated LDH levels. BRAF V600 mutations were observed in 34%. The median time to increase AEC > 400/mm2 was 42 (14-196) days. Pts who experienced an increase > 400/mm3 at 12 weeks demonstrated better outcomes in terms of PFS (21,9 [95% CI: 11,1-32,6] vs. 7,5 [95% CI: 4,3-10,7] months, p=0.017) and OS (38,0 [95% CI: 30,8-45,1] vs. 20,5 [95% CI: 15,7-25,3] months, p=0.025) compared with those who did not. Moreover, according to our previous results, pts with an increase of at least 100/mm3 in AEC over the baseline at 12 weeks also showed better OS (32,5 [95% CI: 26,3-38,8] vs. 16,3 [95% CI: 11,1-21,6] months, p=0.008). Conclusions: An increase in AEC of 100/mm3 over baseline and an absolute AEC > 400/mm3 at week 12 during anti-PD1 tmt might identify pts with MM most likely to experience clinical benefit with anti-PD1 Abs which could be relevant for pts management.

The purpose of this prospective cohort study was to assess the feasibility of outpatient treatment in patients with cancer and objectively confirmed pulmonary embolism (PE), and to compare the performance of the different prognostic scales available in this setting. Patients were selected for outpatient management according to a set of exclusion criteria. Outcomes at 30 and 90 days of follow-up included thromboembolic recurrences, major bleeding, and all-cause death. The performance of 4 prognostic scales (Pulmonary Embolism Severity Index, Geneva Prognostic Score, POMPE-C, and Registro Informatizado de Enfermedad Tromboembolica [RIETE registry]) was evaluated. Of 138 patients, 62 (45%) were managed as outpatients. Incidental PE constituted 47% of the sample. Most patients treated at home had an incidentally detected PE (89%). The rate of recurrence and major bleeding events was similar in both groups. Mortality rates were higher for patients admitted to the hospital compared with outpatients at 30 days (18% vs 3%; P=.06) and 90 days (34% vs 10%; P=.001) of follow-up. None of the patients selected for home treatment required further admission because of PE complications. None of the prognostic models developed for symptomatic PE was significantly associated with 30-day mortality. Improved survival outcomes were observed in incidentally detected PEs compared with acute symptomatic events (overall mortality rates, 3.2% vs 18.4%; P=.006). A large proportion of patients with cancer and PE may be safely treated as outpatients, especially those with incidental PE. Cancer-specific prognostic scales including incidental PE should be developed for the optimal management of PE in this setting.

// Aranzazu Fernandez-Martinez 1, 2 , Tomas Pascual 1, 2 , Giuseppe Perrone 3 , Serafin Morales 4 , Juan de la Haba 5 , Milagros Gonzalez-Rivera 6 , Patricia Galvan 1, 2, 7 , Francesca Zalfa 3 , Michela Amato 3 , Lucia Gonzalez 8 , Miquel Prats 9 , Federico Rojo 10 , Luis Manso 11 , Laia Pare 1, 2 , Immaculada Alonso 1 , Joan Albanell 12 , Ana Vivancos 7 , Antonio Gonzalez 13 , Judit Matito 7 , Sonia Gonzalez 14 , Pedro Fernandez 1 , Barbara Adamo 1, 2 , Montserrat Munoz 1, 2 , Margarita Viladot 1, 2 , Carme Font 1, 2 , Francisco Aya 1, 2 , Maria Vidal 1, 2 , Rosalia Caballero 15 , Eva Carrasco 15 , Vittorio Altomare 3 , Giuseppe Tonini 3 , Aleix Prat 1, 2, 7 , Miguel Martin 6 1 Medical Oncology Department, Hospital Clinic of Barcelona, Barcelona, Spain 2 Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain 3 Department of Medicine, Universita Campus Bio-Medico di Roma, Rome, Italy 4 Medical Oncology Deparment, Arnau de Vilanova de Lleida Universitary Hospital, Lleida, Spain 5 Medical Oncology Department, Reina Sofia University Hospital, Cordoba, Spain 6 Medical Oncology Department, Instituto de Investigacion Sanitaria Gregorio Maranon (IISGM), Universidad Complutense, Madrid, Spain 7 Vall d'Hebron Institute of Oncology, Barcelona, Spain 8 Medical Oncology Department, Quiron Hospital, Madrid, Spain 9 Master of Breast Pathology, University of Barcelona, Barcelona, Spain 10 Pathology Department, Fundacion Jimenez Diaz Health Research Institute (IIS-FJD), Madrid, Spain 11 Medical Oncology Department, Doce de Octubre Hospital, Madrid, Spain 12 Medical Oncology Department, Hospital del Mar, Barcelona, Spain 13 Medical Oncology Department, MD Anderson Cancer Center, Madrid, Spain 14 Medical Oncology Department, Mutua de Terrassa Hospital, Barcelona, Spain 15 Spanish Breast Cancer Research Group Grupo Espanol de Investigacion en Cancer de Mama (GEICAM), Madrid, Spain Correspondence to: Miguel Martin, email: mmartin@geicam.org Aleix Prat, email: aprat@vhio.net Keywords: PAM50/Prosigna, breast cancer, Ki67, estrogen receptor-positive/HER2-negative Received: October 19, 2016 Accepted: January 27, 2017 Published: February 27, 2017 ABSTRACT PAM50/Prosigna gene expression-based assay identifies three categorical risk of relapse groups (ROR-low, ROR-intermediate and ROR-high) in post-menopausal patients with estrogen receptor estrogen receptor-positive (ER+)/ HER2-negative (HER2-) early breast cancer. Low risk patients might not need adjuvant chemotherapy since their risk of distant relapse at 10-years is below 10% with endocrine therapy only. In this study, 517 consecutive patients with ER+/HER2- and node-negative disease were evaluated for Ki67 and Prosigna. Most of Luminal A tumors (65.6%) and ROR-low tumors (70.9%) had low Ki67 values (0-10%); however, the percentage of patients with ROR-medium or ROR-high disease within the Ki67 0-10% group was 42.7% (with tumor sizes ≤2 cm) and 33.9% (with tumor sizes > 2 cm). Finally, we found that the optimal Ki67 cutoff for identifying Luminal A or ROR-low tumors was 14%. Ki67 as a surrogate biomarker in identifying Prosigna low-risk outcome patients or Luminal A disease in the clinical setting is unreliable. In the absence of a well-validated prognostic gene expression-based assay, the optimal Ki67 cutoff for identifying low-risk outcome patients or Luminal A disease remains at 14%.