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Purpose: The PI3K signaling pathway is frequently dysregulated in breast cancer, and mutations in PIK3CA are relevant for therapy resistance in HER2-positive (HER2pos) breast cancer. Mutations in exons 9 or 20 may have different impacts on response to neoadjuvant chemotherapy-based treatment regimens., Experimental Design: We investigated PIK3CA mutations in 1,691 patients with early breast cancer who were randomized into four neoadjuvant multicenter trials: GeparQuattro (NCT00288002), GeparQuinto (NCT00567554), GeparSixto (NCT01426880), and GeparSepto (NCT01583426). The role of different PIK3CA exons and hotspots for pathologic complete response (pCR) following neoadjuvant chemotherapy (NACT) and patient survival were evaluated for distinct molecular subgroups and anti-HER2 treatment procedures., Results: A total of 302 patients (17.9%) of the full cohort of 1,691 patients had a tumor with a PIK3CA mutation, with a different prevalence in molecular subgroups: luminal/HER2-negative (HER2neg) 95 of 404 (23.5%), HER2pos 170 of 819 (20.8%), and triple-negative breast cancer 37 of 468 patients (7.9%). We identified the mutations in PIK3CA exon 20 to be linked with worse response to anti-HER2 treatment (OR = 0.507; 95% confidence interval, 0.320-0.802; P = 0.004), especially in hormone receptor-positive HER2-positive breast cancer (OR = 0.445; 95% confidence interval, 0.237-0.837; P = 0.012). In contrast, exon 9 hotspot mutations p.E452K and p.E545K revealed no noteworthy differences in response therapy. Luminal/HER2neg patients show a trend to have worse treatment response when PIK3CA was mutated. Interestingly, patients with residual disease following neoadjuvant treatment had better survival rates when PIK3CA was mutated., Conclusions: The PIK3CA hotspot mutation p.H1047R is associated with worse pCR rates following NACT in HER2pos breast cancer, whereas hotspot mutations in exon 9 seem to have less impact., (©2024 American Association for Cancer Research.)

Purpose: Eftilagimod alpha (efti), a soluble lymphocyte activation gene (LAG-3) protein and MHC class II agonist, enhances innate and adaptive immunity. Active Immunotherapy PAClitaxel (AIPAC) evaluated safety and efficacy of efti plus paclitaxel in patients with predominantly endocrine-resistant, hormone receptor-positive, HER2-negative metastatic breast cancer (ET-resistant HR+ HER2- MBC)., Patients and Methods: Women with HR+ HER2- MBC were randomized 1:1 to weekly intravenous paclitaxel (80 mg/m2) and subcutaneous efti (30 mg) or placebo every 2 weeks for six 4-week cycles, then monthly subcutaneous efti (30 mg) or placebo maintenance. Primary endpoint was progression-free survival (PFS) by blinded independent central review. Secondary endpoints included overall survival (OS), safety/tolerability, pharmacokinetics/pharmacodynamics, and quality of life. Exploratory endpoints included cellular biomarkers., Results: 114 patients received efti and 112 patients received placebo. Median age was 60 years (91.6% visceral disease, 84.1% ET-resistant, 44.2% with previous CDK4/6 inhibitor treatment). Median PFS at 7.3 months was similar for efti and placebo. Median OS was not significantly improved for efti (20.4 vs. 17.5 months; HR, 0.88; P = 0.197) but became significant for predefined exploratory subgroups. EORTC QLQC30-B23 global health status was sustained for efti but deteriorated for placebo. Efti increased absolute lymphocyte, monocyte and secondary target cell (CD4, CD8) counts, plasma IFNγ and CXCL10 levels., Conclusions: Although the primary endpoint, PFS, was not met, AIPAC confirmed expected pharmacodynamic effects and demonstrated excellent safety profile for efti. OS was not significantly improved globally (2.9-month difference), but was significantly improved in exploratory biomarker subgroups, warranting further studies to clarify efti's role in patients with ET-resistant HER2- MBC., (©2023 The Authors; Published by the American Association for Cancer Research.)

Purpose: The GeparX study investigated whether denosumab as add-on treatment to nab-paclitaxel-based neoadjuvant chemotherapy (NACT) with two different schedules (125 mg/m² weekly vs. day 1, 8 every 22 days) may increase pathologic complete response (pCR) rate. The addition of denosumab to NACT did not improve pCR rates as recently published. In this study, we investigated whether receptor activator of nuclear factor-kappa B (RANK) expression, as part of the denosumab target pathway: (i) may retrospectively identify a subgroup of patients with additional clinical benefit of denosumab or (ii) may predict response to nab-paclitaxel NACT., Experimental Design: RANK protein was IHC-stained on pre-therapeutic core biopsies from patients of the GeparX study (n = 667) with the antibody RANK/Envision System HRP (DAB) and was analyzed for the percentage of membranous RANK tumor cell staining (>5% RANKhigh vs. ≤5% RANKlow)., Results: We could not identify any patient subgroup with differential response under denosumab add-on treatment in patients with RANKhigh expression [139/667, 20.8%; OR, 0.86; 95% confidence interval (CI), 0.44-1.68; P = 0.667] or RANKlow expression (528/667 (79.2%) OR, 1.10; 95% CI, 0.78-1.56; P = 0.589; Pinteraction = 0.528). However, the pCR rate was higher in the RANKhigh subgroup compared with RANKlow (50% vs. 39%; OR, 1.52; 95% CI, 1.04-2.21; P = 0.037). RANK expression constituted an independent predictor of response to NACT frequently in patients with luminal-like subtype (HR+/HER2-; OR, 2.98; 95% CI, 1.30-6.79; P = 0.010). No predictive value of RANK expression among the different nab-paclitaxel regimens was observed., Conclusion: We report RANK expression to be an independent predictive biomarker for response to NACT in patients with luminal-like breast cancer., (©2023 American Association for Cancer Research.)

Purpose: Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high)., Patients and Methods: Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method., Results: A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1-6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40-5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12-5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45-1.22 ( P interaction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74-0.86) to 0.83 (95% CI, 0.78-0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49-0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30-0.86)., Conclusions: Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development., (©2021 American Association for Cancer Research.)

Purpose: The companion diagnostic test for trastuzumab has not changed much in the last 25 years. We used high-plex digital spatial profiling to identify biomarkers besides HER2 that can help predict response to trastuzumab in HER2-positive breast cancer., Experimental Design: Fifty-eight protein targets were measured in three different molecularly defined compartments by the NanoString GeoMx Digital Spatial Profiler (DSP) in a tissue microarray containing 151 patients with breast cancer that received adjuvant trastuzumab as part of the Hellenic Cooperative Oncology Group 10/05 clinical trial. Promising candidate biomarkers were orthogonally validated with quantitative immunofluorescence (QIF). RNA-sequencing data from the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation Study (NeoALTTO) were accessed to provide independent cohort validation. Disease-free survival (DFS) was the main outcome assessed. Statistical analyses were performed using a two-sided test (α = 0.05) and multiple testing correction (Benjamini-Hochberg method, FDR < 0.1)., Results: By DSP, high expression of alpha-smooth muscle actin (α-SMA), both in the leukocyte and stromal compartments, was associated with shorter DFS in univariate analysis ( P = 0.002 and P = 0.023, respectively). High α-SMA expression in the stroma was validated by QIF after controlling for estrogen receptor and progesterone receptor status [HR, 3.12; 95% confidence interval (CI), 1.12-8.68; P = 0.029] showing recurrence on trastuzumab in the same cohort. In the NeoALTTO cohort, elevated levels of ACTA2 were predictive for shorter DFS in the multivariate analysis (HR, 3.21; 95% CI, 1.14-9.05; P = 0.027)., Conclusions: This work identifies α-SMA as a novel, easy-to-implement biomarker of resistance to trastuzumab that may be valuable in settings where trastuzumab is combined with other therapies., (©2021 American Association for Cancer Research.)

Purpose: Data are sparse for oral selective estrogen receptor (ER) degraders (SERD) in cancer treatment. The investigational oral SERD LSZ102 was assessed in monotherapy and combination use in a phase I study., Patients and Methods: A phase I, multicenter, open-label dose-escalation study (NCT02734615) of LSZ102 alone (arm A; n = 77) or with ribociclib (arm B; n = 78) or alpelisib (arm C; n = 43) in heavily pretreated adults with histologically confirmed ER-positive breast cancer and prior disease progression. Arm A received LSZ102 200-900 mg/day; arm B, LSZ102 200-600 mg/day plus ribociclib 300-600 mg/day; arm C, LSZ102 300-450 mg/day plus alpelisib 200-300 mg/day. Key outcomes were dose-limiting toxicities (DLT) in the first 28-day treatment cycle, adverse events (AE), laboratory parameters, pharmacokinetics, biopsy ER protein, and investigator-assessed clinical response (RECIST v1.1)., Results: The most common AEs were gastrointestinal. Treatment-related serious AEs occurred in 10% of participants (19/198), mostly in arm C [10/43 (23%)]. DLTs occurred in: arm A, 5% (4/77); arm B, 3% (2/78); and arm C, 19% (8/43). LSZ102 exposure was slightly greater than dose proportional. On-treatment biopsy ER reductions were observed, with a trend toward an LSZ102 dose response. Objective response rates (95% confidence interval) were: arm A, 1.3% (0.0-7.0); arm B, 16.9% (9.3-27.1); and arm C, 7.0% (1.5-19.1), and clinical benefit rates 7.8% (2.9-16.2), 35.1% (24.5-46.8), and 20.9% (10.0-36.0), respectively., Conclusions: LSZ102 was well tolerated alone and with ribociclib and had a manageable safety profile with alpelisib. Preliminary clinical activity was observed in combination use., (©2021 The Authors; Published by the American Association for Cancer Research.)

Purpose: The heterogeneity of response to anti-HER2 agents represents a major challenge in patients with HER2-positive breast cancer. To better understand the sensitivity and resistance to trastuzumab and lapatinib, we investigated the role of copy number aberrations (CNA) in predicting pathologic complete response (pCR) and survival outcomes in the NeoALTTO trial., Experimental Design: The neoadjuvant phase III NeoALTTO trial enrolled 455 patients with HER2-positive early-stage breast cancer. DNA samples from 269 patients were assessed for genome-wide copy number profiling. Recurrent CNAs were found with GISTIC2.0., Results: CNA estimates were obtained for 184 patients included in NeoALTTO. Among those, matched transcriptome and whole-exome data were available for 154 and 181 patients, respectively. A significant association between gene copy number and pCR was demonstrated for ERBB2 amplification. Nevertheless, ERBB2 amplification ceased to be predictive once ERBB2 expression level was considered. GISTIC2.0 analysis revealed 159 recurrent CNA regions. Lower copy number levels of the 6q23-24 locus predicted absence of pCR in the whole cohort and in the estrogen receptor-positive subgroup. 6q23-24 deletion was significantly more frequent in TP53 wild-type (WT) compared with TP53 -mutated, resulting in copy number levels significantly associated with lack of pCR only in the TP53 WT subgroup. Interestingly, a gene-ontology analysis highlighted several immune processes correlated to 6q23-24 copy number., Conclusions: Our analysis identified ERBB2 copy number as well as 6q23-24 CNAs as predictors of response to anti-HER2-based treatment. ERBB2 expression outperformed ERBB2 amplification. The complexity of the 6q23-24 region warrants further investigation., (©2021 American Association for Cancer Research.)

Purpose: In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug., Experimental Design: We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays., Results: We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21-2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00-4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41-2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28-4.51; P = 0.006) with a significant interaction with bevacizumab treatment ( P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected ( P = 0.025)., Conclusions: Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition., (©2020 American Association for Cancer Research.)

Purpose: To investigate the potential of circulating-miRNAs (ct-miRNA) as noninvasive biomarkers to predict the efficacy of single/dual HER2-targeted therapy in the NeoALTTO study., Experimental Design: Patients with plasma samples at baseline (T0) and/or after 2 weeks (T1) of treatment were randomized into training ( n = 183) and testing ( n = 246) sets. RT-PCR-based high-throughput miRNA profiling was employed in the training set. After normalization, ct-miRNAs associated with pathologic complete response (pCR) were identified by univariate analysis. Multivariate logistic regression models were implemented to generate treatment-specific signatures at T0 and T1, which were evaluated by RT-PCR in the testing set. Event-free survival (EFS) according to ct-miRNA signatures was estimated by Kaplan-Meier method and Cox regression model., Results: In the training set, starting from 51 ct-miRNAs associated with pCR, six signatures with statistically significant predictive capability in terms of area under the ROC curve (AUC) were identified. Four signatures were confirmed in the testing set: lapatinib at T0 and T1 [AUC 0.86; 95% confidence interval (CI), 0.73-0.98 and 0.71 (0.55-0.86)], respectively; trastuzumab at T1 (0.81; 0.70-0.92); lapatinib + trastuzumab at T1 (0.67; 0.51-0.83). These signatures were confirmed predictive after adjusting for known variables, including estrogen receptor status. ct-miRNA signatures failed to correlate with EFS. However, the levels of ct-miR-140-5p, included in the trastuzumab signature, were associated with EFS (HR 0.43; 95% CI, 0.22-0.84)., Conclusions: ct-miRNAs discriminate patients with and without pCR after neoadjuvant lapatinib- and/or trastuzumab-based therapy. ct-miRNAs at week two could be valuable to identify patients responsive to trastuzumab, to avoid unnecessary combination with other anti-HER2 agents, and finally to assist deescalating treatment strategies., (©2019 American Association for Cancer Research.)

Purpose: In the neoadjuvant treatment (NAT) setting, dual HER2-targeted therapy is associated with increased pathologic complete response (pCR) rates compared with each therapy alone. Biomarkers allowing to predict treatment response during NAT are needed. We aim to evaluate whether circulating tumor DNA (ctDNA) is associated with response to anti-HER2-targeted therapy., Experimental Design: Plasma DNA collected before NAT, at week 2, and before surgery from patients enrolled in the NeoALTTO trial was assessed using digital PCR for PIK3CA and TP53 mutation detection., Results: A total of 69 of 455 (15.2%) patients had a PIK3CA and/or TP53 mutation detected in the baseline tumor sample and evaluable ctDNA results from baseline samples. CtDNA was detected in 41%, 20%, and 5% patients before NAT, at week 2, and before surgery, respectively. ctDNA detection before NAT was significantly associated with older age and ER-negative status. ctDNA detection before NAT was associated with decreased odds of achieving pCR (OR = 0.15; 95% CI, 0.034-0.7; P = 0.0089), but not with event-free survival (EFS). Analyses for EFS were underpowered. Interestingly, the patients with HER2-enriched subtype tumors and undetectable ctDNA at baseline had the highest pCR rates. In contrast, patients with persistent ctDNA detection at baseline and week 2 had the lowest rate of pCR., Conclusions: ctDNA detection before neoadjuvant anti-HER2 therapies is associated with decreased pCR rates. Interestingly, patients with HER2-enriched tumors and undetectable ctDNA at baseline had the highest pCR rates, therefore appearing as the best candidates for treatment deescalation strategies., (©2019 American Association for Cancer Research.)

Purpose: This study aimed to evaluate a modified EPclin test (mEPclin), a combination of EndoPredict (EP) score, post-neoadjuvant pathologic tumor size and nodal status, for predicting the risk of distance recurrence after neoadjuvant chemotherapy (NACT) in patients with residual estrogen receptor (ER)-positive/HER2-negative breast cancer. We also compared the prognostic power of the mEPclin with that of the CPS-EG score. Experimental Design: A total of 428 formalin-fixed, paraffin-embedded tumor samples from GeparTrio and GeparQuattro studies were evaluated for mRNA expression of eight cancer-related and three reference genes. The mEPclin score was computed using a modified algorithm and predefined cut-off values were used to classify each patient at low or high risk. Primary endpoint was disease-free survival (DFS). Results: A higher continuous mEPclin score was significantly associated with increased risk of relapse [HR, 2.16; 95% confidence interval (CI), 1.86-2.51; P < 0.001] and death (HR, 2.28; 95% CI, 1.90-2.75; P < 0.001). Similarly, patients classified at high risk by dichotomous mEPclin showed significantly poorer DFS and overall survival compared with those at low risk. In contrast with CPS-EG, the mEPclin remained significantly prognostic for DFS in multivariate analysis (HR, 2.13; 95% CI, 1.73-2.63; P < 0.001). Combining CPS-EG and other clinicopathological variables with mEPclin yielded a significant improvement of the prognostic power for DFS versus without mEPclin (c-indices: 0.748 vs. 0.660; P < 0.001). Conclusions: The mEPclin score independently predicted the risk of distance recurrence and provided additional prognostic information to the CPS-EG score to assess more accurately the prognosis after NACT in the luminal non-pCR patient population. Therefore, this approach can be used to select patients for additional post-neoadjuvant therapies. Clin Cancer Res; 24(14); 3358-65. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: This study aimed to evaluate the prognostic impact of circulating tumor cells (CTC) detected in patients with operable or locally advanced breast cancer before and after neoadjuvant therapy (NT) within the clinical trial GeparQuattro. Experimental Design: Data on CTCs enumerated with the CellSearch system were available for 213 and 207 patients before and after NT, respectively. Associations of CTCs with disease-free survival (DFS) and overall survival (OS) were analyzed by nonparametric Kaplan-Meier estimates and parametric Cox regression. Results: After a median follow-up of 67.1 months, the detection of ≥1 CTC/7.5 mL and ≥2 CTCs/7.5 mL before NT was associated with reduced DFS ( P = 0.031 and P < 0.0001, respectively) and OS ( P = 0.0057 and P < 0.0001, respectively), whereas CTCs detected after NT did not correlate with DFS or OS. In parametric univariate and multivariate Cox models, ≥1 CTC/7.5 mL, ≥2 CTCs/7.5 mL, and absolute CTC numbers before NT revealed to be independent prognostic parameters of DFS and OS. CTC-negative patients with pathologic complete response (pCR) exhibited the best prognosis, whereas those with CTCs and less tumor response were at high risk of tumor relapse. In HER2 (ERBB2)-positive and triple-negative patients, ≥2 CTCs/7.5 mL detected before NT also were significantly associated with worse DFS and OS. Conclusions: Detection of CTCs before NT is an independent prognostic factor of impaired clinical outcome, and combined with pCR, it could be helpful to stratify breast cancer patients for therapeutic interventions. Clin Cancer Res; 23(18); 5384-93. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or pertuzumab has been shown to be superior to single-agent trastuzumab. However, a significant fraction of HER2-overexpressing (HER2 + ) breast cancers escape from these drug combinations. In this study, we sought to discover the mechanisms of acquired resistance to the combination of lapatinib + trastuzumab. Experimental Design: HER2 + BT474 xenografts were treated with lapatinib + trastuzumab long-term until resistance developed. Potential mechanisms of acquired resistance were evaluated in lapatinib + trastuzumab-resistant (LTR) tumors by targeted capture next-generation sequencing. In vitro experiments were performed to corroborate these findings, and a novel drug combination was tested against LTR xenografts. Gene expression and copy-number analyses were performed to corroborate our findings in clinical samples. Results: LTR tumors exhibited an increase in FGF3/4/19 copy number, together with an increase in FGFR phosphorylation, marked stromal changes in the tumor microenvironment, and reduced tumor uptake of lapatinib. Stimulation of BT474 cells with FGF4 promoted resistance to lapatinib + trastuzumab in vitro Treatment with FGFR tyrosine kinase inhibitors reversed these changes and overcame resistance to lapatinib + trastuzumab. High expression of FGFR1 correlated with a statistically shorter progression-free survival in patients with HER2 + early breast cancer treated with adjuvant trastuzumab. Finally, FGFR1 and/or FGF3 gene amplification correlated with a lower pathologic complete response in patients with HER2 + early breast cancer treated with neoadjuvant anti-HER2 therapy. Conclusions: Amplification of FGFR signaling promotes resistance to HER2 inhibition, which can be diminished by the combination of HER2 and FGFR inhibitors. Clin Cancer Res; 23(15); 4323-34. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: HER2-positive breast cancer is heterogeneous. Some tumors express mutations, like activating PIK3CA mutations or reduced PTEN expression, that negatively correlate with response to HER2-targeted therapies. In this exploratory analysis, we investigated whether the efficacy of trastuzumab emtansine (T-DM1), an antibody-drug conjugate comprised of the cytotoxic agent DM1 linked to the HER2-targeted antibody trastuzumab, was correlated with the expression of specific biomarkers in the phase III EMILIA study., Experimental Design: Tumors were evaluated for HER2 (n = 866), EGFR (n = 832), and HER3 (n = 860) mRNA expression by quantitative reverse transcriptase PCR; for PTEN protein expression (n = 271) by IHC; and for PIK3CA mutations (n = 259) using a mutation detection kit. Survival outcomes were analyzed by biomarker subgroups. T-DM1 was also tested on cell lines and in breast cancer xenograft models containing PIK3CA mutations., Results: Longer progression-free survival (PFS) and overall survival (OS) were observed with T-DM1 compared with capecitabine plus lapatinib in all biomarker subgroups. PIK3CA mutations were associated with shorter median PFS (mutant vs. wild type: 4.3 vs. 6.4 months) and OS (17.3 vs. 27.8 months) in capecitabine plus lapatinib-treated patients, but not in T-DM1-treated patients (PFS, 10.9 vs. 9.8 months; OS, not reached in mutant or wild type). T-DM1 showed potent activity in cell lines and xenograft models with PIK3CA mutations., Conclusions: Although other standard HER2-directed therapies are less effective in tumors with PI3KCA mutations, T-DM1 appears to be effective in both PI3KCA-mutated and wild-type tumors. Clin Cancer Res; 22(15); 3755-63. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: The PI3K/AKT pathway and phosphatase and tensin homolog (PTEN) aberrations are common in breast cancer. We investigated the correlation between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), PTEN, p4EBP1 (phosphorylated E4 binding protein 1), and pathologic complete response (pCR) in patients receiving neoadjuvant therapy., Experimental Design: We retrospectively evaluated PIK3CA, PTEN, and p4EBP1 protein expression in centrally HER2-positive patients (n = 181) who received epirubicin cyclophosphamide/trastuzumab followed by docetaxel/trastuzumab alone or concomitant/followed by capecitabine within the GeparQuattro study. PTEN was assessed using the automated quantitative immunofluorescence analysis and was analyzed as a dichotomic variable. p4EBP1 was assessed by immunohistochemistry and used as a continuous and dichotomic variable., Results: p4EBP1 was available from 137, PTEN from 108, and PIK3CA genotype from 83 patients. Overall, the pCR rate in PTEN-low tumors was 27.6%, and in PTEN-high tumors, it was 57.1% (P = 0.010). pCR rates were not statistically different between PIK3CA wild-type and mutant (35% vs. 22%) or p4EBP1 IRS ≤ 4 and IRS > 4 (39% vs. 33%). pCR rate was 57.1% (8/14) in PTEN-high/PIK3CA wild-type and decreased to 15.4% in PTEN-low/PIK3CA-mutant tumors (P = 0.023). In multivariable analysis adjusted for baseline parameters, PTEN independently predicted pCR in the following cohorts: overall [OR, 7.54; 95% confidence interval (CI), 2.03-28.06; P = 0.003], PIK3CA wild-type (OR, 23.81; 95% CI, 1.75-324.05; P = 0.017), p4EBP1 IRS > 4 (OR, 11.53; 95% CI, 1.84-72.24; P = 0.009), and hormone receptor-positive (OR, 40.91; 95% CI, 2.93-570.44; P = 0.006). p4EBP1 was independently predictive for pCR in PIK3CA wild-type tumors (OR, 0.14; 95% CI, 0.03-0.78; P = 0.025)., Conclusions: The study showed the potential role of PIK3CA genotype, PTEN, and p4EBP in predicting pCR after anthracycline-taxane-based chemotherapy and anti-HER2 treatment. Clin Cancer Res; 22(11); 2675-83. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: Scoring proliferation through Ki67 immunohistochemistry is an important component in predicting therapy response to chemotherapy in patients with breast cancer. However, recent studies have cast doubt on the reliability of "visual" Ki67 scoring in the multicenter setting, particularly in the lower, yet clinically important, proliferation range. Therefore, an accurate and standardized Ki67 scoring is pivotal both in routine diagnostics and larger multicenter studies., Experimental Design: We validated a novel fully automated Ki67 scoring approach that relies on only minimal a priori knowledge on cell properties and requires no training data for calibration. We applied our approach to 1,082 breast cancer samples from the neoadjuvant GeparTrio trial and compared the performance of automated and manual Ki67 scoring., Results: The three groups of autoKi67 as defined by low (≤ 15%), medium (15.1%-35%), and high (>35%) automated scores showed pCR rates of 5.8%, 16.9%, and 29.5%, respectively. AutoKi67 was significantly linked to prognosis with overall and progression-free survival P values P(OS) < 0.0001 and P(PFS) < 0.0002, compared with P(OS) < 0.0005 and P(PFS) < 0.0001 for manual Ki67 scoring. Moreover, automated Ki67 scoring was an independent prognosticator in the multivariate analysis with P(OS) = 0.002, P(PFS) = 0.009 (autoKi67) versus P(OS) = 0.007, PPFS = 0.004 (manual Ki67)., Conclusions: The computer-assisted Ki67 scoring approach presented here offers a standardized means of tumor cell proliferation assessment in breast cancer that correlated with clinical endpoints and is deployable in routine diagnostics. It may thus help to solve recently reported reliability concerns in Ki67 diagnostics., (©2014 American Association for Cancer Research.)

Purpose: Dual anti-HER2 blockade with trastuzumab/pertuzumab or trastuzumab/lapatinib in combination with anthracycline/taxane-based chemotherapy can reach pathologic complete response (pCR) rates of up to 60% in HER2-positive breast cancer. The DAFNE (Dual blockade with AFatinib and trastuzumab as NEoadjuvant treatment) phase II study (NCT015591477) investigated a dual blockade with the irreversible pan-HER inhibitor afatinib and trastuzumab in this setting., Experimental Design: Participants with untreated, centrally HER2-positive breast cancer were treated for 6 weeks with afatinib (20 mg/d) and trastuzumab [(8) 6 mg/kg/3 weeks] alone; followed by 12-week treatment with paclitaxel (80 mg/m(2)/1 week), trastuzumab, and afatinib; followed by 12 weeks with epirubicin (90 mg/m(2)/3 weeks), cyclophosphamide (600 mg/m(2)/3 weeks), and trastuzumab before surgery. Primary objective was pCR rate, defined as ypT0/is ypN0. We expected a pCR rate of 70%; 65 patients were needed to exclude a rate of ≤55%., Results: pCR rate was 49.2% [90% confidence interval (CI), 38.5-60.1] in 65 treated patients. Patients with hormone receptor-negative (N = 19) or hormone receptor-positive (N = 46) tumors showed pCR rates of 63.2% and 43.5%, respectively (P = 0.153). Patients with (N = 9) or without (N = 56) lymphocyte predominant breast cancer (LPBC) showed pCR rates of 100% and 41.1%, respectively (P < 0.001). PCR rate was not different in patients with or without PIK3CA tumor mutations (P = 0.363). Clinical responses were seen in 96.3% of 54 evaluable patients, and breast conserving surgery was possible in 59.4% of 62 assessable patients. Most frequent nonhematologic grade 3-4 toxicities were diarrhea (7.7%), increased creatinine (4.6%), and infection (4.6%). One patient developed symptomatic congestive heart failure., Conclusions: Neoadjuvant treatment with afatinib, trastuzumab, and chemotherapy showed acceptable tolerability, and a pCR rate comparable with that of other anti-HER2 doublets but below challenging expectations., (©2015 American Association for Cancer Research.)

Purpose: The value of Ki67 measured on residual disease after neoadjuvant chemotherapy is not sufficiently described., Experimental Design: Participants of the GeparTrio study with primary breast cancer randomly received neoadjuvant response-guided [8 cycles TAC (docetaxel/doxorubicin/cyclophosphamide) in responding and TAC-NX (vinorelbine/capecitabine) in nonresponding patients] or conventional (6 cycles TAC) chemotherapy according to interim response assessment. Ki-67 levels were centrally measured immunohistochemically after neoadjuvant treatment if tumor tissue was available. Here, we analyze 1,151 patients having a pathologic complete response (pCR; n, 484), or residual disease with low (0-15%), intermediate (15.1-35%), or high (35.1-100%) posttreatment Ki67 levels in 488, 77, and 102 patients, respectively., Results: Patients with high posttreatment Ki67 levels showed higher risk for disease relapse (P < 0.0001) and death (P < 0.0001) compared with patients with low or intermediate Ki67 levels. Patients with low Ki67 levels showed a comparable outcome to patients with a pCR (P = 0.211 for disease-free and P = 0.779 for overall survival). Posttreatment Ki67 levels provided more prognostic information than pretreatment Ki67 levels or changes of Ki67 from pre- to posttreatment. Information on pCR plus posttreatment Ki67 levels surmount the prognostic information of pCR alone in hormone-receptor-positive disease [hazard ratios (HR), 1.82-5.88] but not in hormone-receptor-negative disease (HR: 0.61-1.73). Patients with conventional and response-guided treatment did not show a different distribution of posttreatment Ki67 (P = 0.965)., Conclusions: Posttreatment Ki67 levels provide prognostic information for patients with hormone-receptor-positive breast cancer and residual disease after neoadjuvant chemotherapy. Levels were not prognostic for outcome after response-guided chemotherapy. High posttreatment Ki67 indicates the need for innovative postneoadjuvant treatments., (©2013 AACR.)

Purpose: This study was aimed at detecting and characterizing circulating tumor cells (CTC) before and after neoadjuvant therapy (NT) in the peripheral blood of patients with breast cancer., Experimental Design: The clinical trial GeparQuattro incorporated NT approaches (epirubicin/cyclophosphamide prior to randomization to docetaxel alone, docetaxel in combination with capecitabine, or docetaxel followed by capecitabine) and additional trastuzumab treatment for patients with HER2-positive tumors. We used the Food and Drug Administration-approved CellSearch system for CTC detection and evaluation of HER2 expression and developed HER2 immunoscoring for CTC., Results: We detected > or =1 CTC/7.5 mL in 46 of 213 patients (21.6%) before NT and in 22 of 207 patients (10.6%) after NT (P = 0.002). Twenty (15.0%) initially CTC-positive cases were CTC-negative after NT, whereas 11 (8.3%) cases were CTC-positive after NT, although no CTC could be found before NT. CTC detection did not correlate with primary tumor characteristics. Furthermore, there was no association between tumor response to NT and CTC detection. HER2-overexpressing CTC were observed in 14 of 58 CTC-positive patients (24.1%), including 8 patients with HER2-negative primary tumors and 3 patients after trastuzumab treatment. CTC scored HER2-negative or weakly HER2-positive before or after NT were present in 11 of 21 patients with HER2-positive primary tumors. HER2 overexpression on CTC was restricted to ductal carcinomas and associated with high tumor stage (P = 0.002)., Conclusion: CTC number was low in patients with primary breast cancer. The decrease in CTC incidence during treatment was not correlated with standard clinical characteristics and primary tumor response. Information on the HER2 status of CTC might be helpful for stratification and monitoring of HER2-directed therapies., (Copyright 2010 AACR.)

Purpose: A Phase I/IIb multicenter study was conducted to evaluate the safety and immunogenicity of the anti-idiotypic antibody vaccine ACA125 that functionally imitates the tumor antigen CA125 in 119 patients with advanced ovarian carcinoma. A preliminary report on the initial 42 patients demonstrated safety and immunogenicity., Experimental Design: Using the complete intention-to-treat population (n = 119) who received a mean of 9.7 ACA125 applications, survival was analyzed with respect to immunological responses., Results: In 81 patients (68.1%), a specific anti-anti-idiotypic antibody (Ab3) response could be induced. Additionally, the development of CA125-specific antibodies (Ab1') and antibody-dependent cell-mediated cytotoxicity of CA125-positive tumor cells was observed in 50.4% and 26.9% of patients, respectively. The median survival of all patients was 19.4 months (range, 0.5-56.1 months). Ab3-positive patients showed a significantly longer survival (median, 23.4 months; P < 0.0001) as compared with Ab3-negative patients (median, 4.9 months). A positive Ab3 response remained associated with longer survival when controlling for other prognostic factors including FIGO (International Federation of Gynecologists and Obstetricians) stage, response to and type of first-line chemotherapy, number of previous treatments, or concomitant antitumor therapy. With regard to safety, repeated vaccination was well tolerated. No serious adverse events related to the application of ACA125 occurred., Conclusions: Although the uncontrolled design of this study prevents definitive conclusions with respect to subgroups, the data support a relationship between Ab3 response and survival time. Thus, the need for further randomized, controlled clinical trials to establish efficacy of the vaccine ACA125 seems to be indicated.

The aim of the present study was to assess whether the induction of specific immune responses by vaccination with the murine monoclonal anti-idiotypic antibody ACA125, which imitates the tumor-associated antigen CA125, has a positive influence on the survival of patients with recurrent ovarian carcinoma. Forty-two patients with platinum-pretreated recurrences were included in a clinical Phase I/II trial of consolidation in third-line therapy. Patients initially received four immunizations with 2 mg of alum-precipitated anti-idiotype ACA125 every 2 weeks and then monthly applications. No serious allergic reactions could be detected within a maximal control period of 56 months. Hyperimmune sera of 27 of 42 patients (64.2%) showed increased concentrations of human antimouse antibodies. Specific anti-anti-idiotypic antibodies as a marker for induced immunity were detected in 28 of 42 patients (66.7%). The survival of the whole ACA125-treated collective of patients after a mean of 12.6 antibody applications was 14.9 +/- 12.9 months. The survival of patients with a positive immune response was 19.9 +/- 13.1 months in contrast with 5.3 +/- 4.3 months in those patients without detectable anti-CA125 immunity (P < 0.0001). According to these results, vaccination with a suitable anti-idiotypic antibody offers an effective way to induce specific immunity against a primarily nonimmunogenic tumor antigen such as CA125 and is associated with a positive impact on the survival of patients with recurrent ovarian cancer with few side effects, which warrants a Phase III trial for ovarian cancer patients after primary therapy.