Differential immunomodulatory effects of epirubicin/cyclophosphamide and docetaxel in breast cancer patients.

BACKGROUND: Epirubicin/cyclophosphamide (EC) and docetaxel (D) are commonly used in a sequential regimen in the neoadjuvant treatment of early, high-risk or locally advanced breast cancer (BC). Novel approaches to increase the response rate combine this treatment with immunotherapies such as PD-1 inhibition. However, the expected stimulatory effect on lymphocytes may depend on the chemotherapy backbone. Therefore, we separately compared the immunomodulatory effects of EC and D in the setting of a randomized clinical trial. METHODS: Tumor and blood samples of 154 patients from the ABCSG-34 trial were available (76 patients received four cycles of EC followed by four cycles of D; 78 patients get the reverse treatment sequence). Tumor-infiltrating lymphocytes, circulating lymphocytes and 14 soluble immune mediators were determined at baseline and at drug change. Furthermore, six BC cell lines were treated with E, C or D and co-cultured with immune cells. RESULTS: Initial treatment with four cycles of EC reduced circulating B and T cells by 94% and 45%, respectively. In contrast, no comparable effects on lymphocytes were observed in patients treated with initial four cycles of D. Most immune mediators decreased under EC whereas D-treatment resulted in elevated levels of CXCL10, urokinase-type plasminogen activator (uPA) and its soluble receptor (suPAR). Accordingly, only the exposure of BC cell lines to D induced similar increases as compared to E. While treatment of BC cells with E was associated with cell shrinkage and apoptosis, D induced cell swelling and accumulation of cells in G2 phase. CONCLUSION: The deleterious effect of EC on lymphocytes indicates strong immunosuppressive properties of this combination therapy. D, in contrast, has no effect on lymphocytes, but triggers the secretion of stimulatory proteins in vivo and in vitro, indicating a supportive effect on the immune system. Underlying differences in the induced cell death might be causal. These divergent immunomodulatory effects of epirubicin/cyclophosphamide and docetaxel should be considered when planning future combinations with immunotherapies in breast cancer.

Estrogen Receptor Alpha Gene Amplification Is an Independent Predictor of Long-Term Outcome in Postmenopausal Patients with Endocrine-Responsive Early Breast Cancer.

PURPOSE: Estrogen receptor (ER) expression is a prognostic parameter in breast cancer, and a prerequisite for the use of endocrine therapy. In ER+ early breast cancer, however, no receptor-associated biomarker exists that identifies patients with a particularly favorable outcome. We have investigated the value of ESR1 amplification in predicting the long-term clinical outcome in tamoxifen-treated postmenopausal women with endocrine-responsive breast cancer. EXPERIMENTAL DESIGN: 394 patients who had been randomized into the tamoxifen-only arm of the prospective randomized ABCSG-06 trial of adjuvant endocrine therapy with available formalin-fixed, paraffin-embedded tumor tissue were included in this analysis. IHC ERα expression was evaluated both locally and in a central lab using the Allred score, while ESR1 gene amplification was evaluated by FISH analysis using the ESR1/CEP6 ratio indicating focal copy number alterations. RESULTS: Focal ESR1 copy-number elevations (amplifications) were detected in 187 of 394 (47%) tumor specimens, and were associated with a favorable outcome: After a median follow-up of 10 years, women with intratumoral focal ESR1 amplification had a significantly longer distant recurrence-free survival [adjusted HR, 0.48; 95% confidence interval (CI), 0.26-0.91; P = 0.02] and breast cancer-specific survival (adjusted HR 0.47; 95% CI, 0.27-0.80; P = 0.01) as compared with women without ESR1 amplification. IHC ERα protein expression, evaluated by Allred score, correlated significantly with focal ESR1 amplification (P < 0.0001; χ2 test), but was not prognostic by itself. CONCLUSIONS: Focal ESR1 amplification is an independent and powerful predictor for long-term distant recurrence-free and breast cancer-specific survival in postmenopausal women with endocrine-responsive early-stage breast cancer who received tamoxifen for 5 years.

Persistence of ctDNA in Patients with Breast Cancer During Neoadjuvant Treatment Is a Significant Predictor of Poor Tumor Response.

PURPOSE: Accurate response assessment during neoadjuvant systemic treatment (NST) poses a clinical challenge. Therefore, a minimally invasive assessment of tumor response based on cell-free circulating tumor DNA (ctDNA) may be beneficial to guide treatment decisions. EXPERIMENTAL DESIGN: We profiled 93 genes in tissue from 193 patients with early breast cancer. Patient-specific assays were designed for 145 patients to track ctDNA during NST in plasma. ctDNA presence and levels were correlated with complete pathological response (pCR) and residual cancer burden (RCB) as well as clinicopathologic characteristics of the tumor to identify potential proxies for ctDNA release. RESULTS: At baseline, ctDNA could be detected in 63/145 (43.4%) patients and persisted in 25/63 (39.7%) patients at mid-therapy (MT) and 15/63 (23.8%) patients at the end of treatment. ctDNA detection at MT was significantly associated with higher RCB (OR = 0.062; 95% CI, 0.01-0.48; P = 0.0077). Of 31 patients with detectable ctDNA at MT, 30 patients (96.8%) were nonresponders (RCB II, n = 8; RCB III, n = 22) and only one patient responded to the treatment (RCB I). Considering all 145 patients with baseline (BL) plasma, none of the patients with RCB 0 and only 6.7% of patients with RCB I had ctDNA detectable at MT, whereas 30.6% and 29.6% of patients with RCB II/III, respectively, had a positive ctDNA result. CONCLUSIONS: Overall, our results demonstrate that the detection and persistence of ctDNA at MT may have the potential to negatively predict response to neoadjuvant treatment and identify patients who will not achieve pCR or be classified with RCB II/III.

The OncoMasTR Test Predicts Distant Recurrence in Estrogen Receptor-Positive, HER2-Negative Early-Stage Breast Cancer: A Validation Study in ABCSG Trial 8.

PURPOSE: To validate the clinical performance of the OncoMasTR Risk Score in the biomarker cohort of Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 8. EXPERIMENTAL DESIGN: We evaluated the OncoMasTR test in 1,200 formalin-fixed, paraffin-embedded (FFPE) surgical specimens from postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative primary breast cancer with 0 to 3 involved lymph nodes in the prospective, randomized ABCSG Trial 8. Time to distant recurrence (DR) was analyzed by Cox models. RESULTS: The OncoMasTR Risk Score categorized 850 of 1,087 (78.2%) evaluable patients as "low risk". At 10 years, the DR rate for patients in the low-risk group was 5.8% versus 21.1% for patients in the high-risk group (P < 0.0001, absolute risk reduction 15.3%). The OncoMasTR Risk Score was highly prognostic for prediction of DR in years 0 to 10 in all patients [HR 1.91, 95% confidence interval (CI) 1.62-2.26, P < 0.0001; C-index 0.73], in patients that were node negative (HR 1.79, 95% CI, 1.43-2.24, P < 0.0001; C-index 0.72), and in patients with 1 to 3 involved lymph nodes (HR 1.93, 95% CI, 1.44-2.58, P < 0.0001; C-index 0.71). The OncoMasTR Risk Score provided significant additional prognostic information beyond clinical parameters, Ki67, Nottingham Prognostic Index, and Clinical Treatment Score. CONCLUSIONS: OncoMasTR Risk Score is highly prognostic for DR in postmenopausal women with ER-positive, HER2-negative primary breast cancer with 0 to 3 involved lymph nodes. In combination with prior validation studies, this fully independent validation in ABCSG Trial 8 provides level 1B evidence for the prognostic capability of the OncoMasTR Risk Score.

Conventional versus reverse sequence of neoadjuvant epirubicin/cyclophosphamide and docetaxel: sequencing results from ABCSG-34.

BACKGROUND: Preoperative chemotherapy containing anthracyclines and taxanes is well established in early-stage breast cancer. Previous studies have suggested that the chemotherapy sequence may matter but definitive evidence is missing. ABCSG trial 34 evaluated the activity of the MUC1 vaccine tecemotide when added to neoadjuvant treatment; the study provided the opportunity for the second randomisation to compare two different anthracycline/taxane sequences. METHODS: HER2-negative early-stage breast cancer patients were recruited to this randomised multicentre Phase 2 study. Patients in the chemotherapy cohort (n = 311) were additionally randomised to a conventional or reversed sequence of epirubicin/cyclophosphamide and docetaxel. Residual cancer burden (RCB) with/without tecemotide was defined as primary study endpoint; RCB in the two chemotherapy groups was a key secondary endpoint. RESULTS: No significant differences in terms of RCB 0/I (40.1% vs. 37.2%; P = 0.61) or pathologic complete response (pCR) rates (24.3% vs. 25%, P = 0.89) were observed between conventional or reverse chemotherapy sequence. No new safety signals were reported, and upfront docetaxel did not result in decreased rates of treatment delay or discontinuation. CONCLUSION: Upfront docetaxel did not improve chemotherapy activity or tolerability; these results suggest that upfront neoadjuvant treatment with anthracyclines remains a valid option.

A risk stratification algorithm for lesions of uncertain malignant potential diagnosed by vacuum-assisted breast biopsy (VABB) of mammographic microcalcifications.

PURPOSE: To investigate a risk stratification strategy for lesions of uncertain malignant potential (B3) diagnosed by vacuum-assisted breast biopsy (VABB) of mammographic microcalcifications. METHODS: Patients who underwent VABB for microcalcification-only lesions with a diagnosis of B3 and subsequent surgery were included in this retrospective, IRB-approved study. Seventy-six B3-lesions (final histology: 66 benign, 10 malignant) were included (Tr). Data on B3 lesion type and presence of atypia, microcalcification characteristics (BI-RADS), removal at biopsy and concomitant lesions were collected. After univariate analysis (Chi-square test), data were combined into a risk stratification algorithm by using a ten-fold, cross-validated Classification and Regression Tree analysis (CRT). The algorithm was tested on a testing dataset (Te) of 23 B3-lesions (six malignant, 17 benign). RESULTS: Malignancy was more frequent in women with a concomitant cancer (P < 0.001) and highly suspicious microcalcifications (P < 0.001). The CRT algorithm retained three characteristics: morphology; presence of atypia; presence of concomitant cancer. The algorithm identified 25/76 (32.9 %,Tr) and 6/23 (26.1 %,Te) lesions at low risk of malignancy. No malignant cases were identified at surgery (0/31). There were 3/76 (3.9 %,Tr) and 1/23 (4.3 %,Te) lesions assigned as high-risk by the algorithm and confirmed at surgery (4/4). In the remaining lesions (48/76, 63.1 %,Tr; 16/23, 69.6 %,Te), malignancy rates varied between 9% and 88.4 %; thus, surgery could not have been avoided. CONCLUSION: We constructed and tested a risk stratification algorithm for B3 microcalcifications, including clinical, imaging, and pathological features, to assign probabilities of malignancy, which has the potential to reduce unnecessary surgeries.

Predictive Value of Molecular Subtypes in Premenopausal Women with Hormone Receptor-positive Early Breast Cancer: Results from the ABCSG Trial 5.

PURPOSE: To assess the predictive value of molecular breast cancer subtypes in premenopausal patients with hormone receptor-positive early breast cancer who received adjuvant endocrine treatment or chemotherapy. EXPERIMENTAL DESIGN: Molecular breast cancer subtypes were centrally assessed on whole tumor sections by IHC in patients of the Austrian Breast and Colorectal Cancer Study Group Trial 5 who had received either 5 years of tamoxifen/3 years of goserelin or six cycles of cyclophosphamide, methotrexate, and fluorouracil (CMF). Luminal A disease was defined as Ki67 <20% and luminal B as Ki67 ≥20%. The luminal B/HER2-positive subtype displayed 3+ HER2-IHC or amplification by ISH. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using Cox models adjusted for clinical and pathologic factors. RESULTS: 185 (38%), 244 (50%), and 59 (12%) of 488 tumors were classified as luminal A, luminal B/HER2-negative and luminal B/HER2-positive, respectively. Luminal B subtypes were associated with poor outcome. Patients with luminal B tumors had a significantly shorter RFS [adjusted HR for recurrence: 2.22; 95% confidence interval (CI), 1.41-3.49; P = 0.001] and OS (adjusted HR for death: 3.51; 95% CI, 1.80-6.87; P < 0.001). No interaction between molecular subtypes and treatment was observed (test for interaction: P = 0.84 for RFS; P = 0.69 for OS). CONCLUSIONS: Determination of molecular subtypes by IHC is an independent prognostic factor for recurrence and death in premenopausal women with early-stage, hormone receptor-positive breast cancer but is not predictive for outcome of adjuvant treatment with tamoxifen/goserelin or CMF.See related commentary by Hunter et al., p. 5543.

The EndoPredict score predicts response to neoadjuvant chemotherapy and neoendocrine therapy in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer patients from the ABCSG-34 trial.

BACKGROUND: Neoadjuvant chemotherapy (NaCT) and neoadjuvant endocrine therapy (NET) can reduce pre-operative tumour burden in patients with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative early-stage breast cancer. This prospective translational study assessed the ability of a 12-gene molecular score (MS; EndoPredict®) to predict response to NaCT or NET within the ABCSG-34 trial. PATIENTS AND METHODS: Hormone receptor (HR)-positive, HER2-negative samples from patients in the ABCSG-34 randomized phase II trial were selected and EndoPredict testing was performed to generate a 12-gene MS. ABCSG-34 patients were assigned to receive either NaCT or NET based on menopausal status, HR expression, grade and Ki67. Response was measured by residual cancer burden (RCB). RESULTS: Patients selected for NaCT generally had high-risk disease by 12-gene MS (125/134), while slightly more patients treated with NET had low-risk disease (44/83). Low-risk NaCT-treated and high-risk NET-treated tumours responded poorly (NPV 100% [95% CI 66.4%-100%] and NPV 92.3% [95% CI 79.1%-98.4%], respectively]. The 12-gene MS significantly predicted treatment response for NaCT (AUC 0.736 [95% CI 0.63-0.84]) and NET (AUC 0.726 [95% CI 0.60-0.85]). CONCLUSIONS: The 12-gene MS predicted RCB after treatment with neoadjuvant therapies for patients with HR-positive, HER2-negative early-stage breast cancer. Tumours with low MS were unlikely to benefit from NaCT, whereas a high MS predicted resistance to NET. This additional biologic information can aid personalized treatment selection in daily practice and builds a strong rationale to use EndoPredict in biomarker-driven studies in the neoadjuvant setting.

Prognostic Value of EndoPredict in Women with Hormone Receptor-Positive, HER2-Negative Invasive Lobular Breast Cancer.

PURPOSE: Invasive lobular carcinoma (ILC) accounts for approximately 5%-15% of all invasive breast cancer cases. Most of the correlations between multigene assays and patient outcome were derived from studies based on patients with invasive ductal carcinoma (IDC) or without distinction between the subtypes. Here, we investigate the prognostic value of EndoPredict (EPclin) in a large cohort of ILCs pooled from three phase III randomized trials (ABCSG-6, ABCSG-8, TransATAC). EXPERIMENTAL DESIGN: The primary objective of this analysis was to determine the prognostic value of EPclin for distant recurrence (DR) in years 0-10 in postmenopausal women with ILC. The primary outcome was DR. RESULTS: 470 women (17.9%) presented with ILC, 1,944 (73.9%) with IDC, and 216 (8.2%) with other histologic types. EPclin was highly prognostic in women with ILC [HR = 3.32 (2.54-4.34)] and provided more prognostic value than the Clinical Treatment Score [CTS; HR = 2.17 (1.73-2.72)]. 63.4% of women were categorized into the low EPclin risk group and they had a 10-year DR of 4.8% (2.7-8.4) compared with 36.6% of women in the high-risk group with a 10-year DR risk of 26.6% (20.0-35.0). EPclin also provided highly prognostic information in women with node-negative disease [HR = 2.56 (1.63-4.02)] and node-positive disease [HR = 3.70 (2.49-5.50)]. CONCLUSIONS: EPclin provided highly significant prognostic value and significant risk stratification for women with ILC. Ten-year DR risk in the EPclin low-risk groups were similar between ILC and IDC. Our results show that EPclin is informative in women with ILC and suggest that it is equally valid in both histologic subtypes.

Efficacy and safety of the therapeutic cancer vaccine tecemotide (L-BLP25) in early breast cancer: Results from a prospective, randomised, neoadjuvant phase II study (ABCSG 34).

BACKGROUND: Immune-based strategies represent a promising approach in breast cancer (BC) treatment. The glycoprotein mucin-1 (MUC-1) is overexpressed in more than 90% of BC patients, and is targeted by the cancer vaccine tecemotide. We have investigated the efficacy and safety of tecemotide when added to neoadjuvant standard-of-care (SoC) treatment in early BC patients. PATIENTS AND METHODS: A total of 400 patients with HER2-early BC were recruited into this prospective, multicentre, randomised 2-arm academic phase II trial. Patients received preoperative SoC treatment (chemotherapy or endocrine therapy) with or without tecemotide. Postmenopausal women with oestrogen receptor (ER)+++, or ER++ and Ki67 < 14%, and G1,2 tumours ('luminal A' tumours) received 6 months of letrozole. Postmenopausal patients with triple-negative, ER-/+/++ and Ki67 ≥ 14%, and with G3 tumours, as well as premenopausal patients, received four cycles of epirubicin/cyclophosphamide plus four cycles of docetaxel. Primary end-point was residual cancer burden (RCB; 0/I versus II/III) at surgery. Secondary end-points included pathological complete response (pCR), safety, and quality of life. FINDINGS: We observed no significant difference in RCB 0/I rates between patients with (36.4%) and without (31.9%) tecemotide in the overall study population (p = 0.40) nor in endocrine and chemotherapy-treated subgroups (25.0% versus 13.3%, p = 0.17; 39.6% versus 37.8%, p = 0.75, respectively). The addition of tecemotide did not affect overall pCR rates (22.5% versus 17.4%, p = 0.23), MUC-1 expression, or tumour-infiltrating lymphocytes content. Tecemotide did not increase toxicity when compared to SoC therapy alone. INTERPRETATION: Neoadjuvant tecemotide is safe, but does not improve RCB or pCR rates in patients receiving standard neoadjuvant therapy.

Association of Cytokeratin 5 and Claudin 3 expression with BRCA1 and BRCA2 germline mutations in women with early breast cancer.

BACKGROUND: It is important to identify biomarkers associated with BRCA mutation in women with early breast cancer (BC) to improve early identification of mutation carriers. Thus, in this study, we examined the protein expression of claudin (CLDN) 3, CLDN4, CLDN7, and E-cadherin. Moreover, we analyzed additional histopathological variables and their associations in familial BC. METHODS: Immunohistochemical analysis for CLDNs and E-cadherin was performed on 237 BC cases of three different subsets of BC tumors: 62 from BRCA1 mutation carriers, 59 from BRCA2 mutation carriers, and 116 tumors from patients with BRCA wild type (WT) as controls. Histopathological data were also analyzed in the different subgroups. Logistic regression and receiver operation characteristic (ROC) curve were conducted to investigate factors associated with BRCA tumors. RESULTS: Expression of CLDN3 positively correlated with BRCA-mutated BC. CLDN3 was expressed in 58% of BRCA1-mutated tumors compared to only 7% in BRCA2-mutated tumors (p < 0.001) and 1% in WT tumors (p < 0.001). CK5 and CK14 expression were also more likely to arise in BRCA1 tumors (44 and 16%, respectively) than in the control group (8 and 4%) (p < 0.001, p = 0.012, respectively). We also found a significantly higher proportion of CK5+ among BRCA1 tumors (44%) in comparison with BRCA2-related BC (8%) (p < 0.001). In addition, there was a significant difference between both groups regarding CK14: positive expression in 16 and 5%, respectively (p = 0.030). CK5 and CK14 did not differ between the BRCA2 group and the WT tumors significantly. In a multivariate regression model, expression of CK5 (Odds ratio (OR): 6.46; 95% confidence interval (CI): 1.52-27.43; p = 0.011), and CLDN3 (OR: 200.48; 95% CI: 21.52-1867.61; p < 0.001) were associated with BRCA1 mutation status. CONCLUSIONS: Our data suggests that CLDN3, CK5, and CK14 in combination with ER, PR and HER2 are associated with BRCA1 mutation status.

Prediction of Distant Recurrence Using EndoPredict Among Women with ER+, HER2- Node-Positive and Node-Negative Breast Cancer Treated with Endocrine Therapy Only.

PURPOSE: Prognostic molecular assays may aid in treatment decisions for women with estrogen receptor (ER)-positive, HER2-negative breast cancer. The prognostic value of a 12-gene expression assay (EndoPredict) was reevaluated in the combined ABCSG-6/8 cohorts with longer clinical follow-up. EXPERIMENTAL DESIGN: EndoPredict (EP; molecular score, EPclin score) was evaluated in women with ER-positive, HER2-negative node-positive and node-negative breast cancer who received 5 years of endocrine therapy only (median follow-up, 9.6 years; N = 1,702). Distant recurrence-free rate (DRFR; 95% confidence interval) was assessed 10 and 15 years after diagnosis. RESULTS: Overall, 62.6% of patients had low-risk EPclin scores with significantly improved DRFR relative to high-risk patients (HR, 4.77; 95% CI, 3.37-6.67; P < 0.0001). Ten-year DRFR (0-10 years) was improved among patients with low-risk versus high-risk EPclin scores in the full cohort [95.5% (94.1%-97.0%) vs. 80.3% (76.9%-83.9%)] as well as for patients with node-negative disease [95.5% (94.0%-97.1%) vs. 87.0% (82.6%-91.7%)] or with 1 to 3 positive nodes [95.6% (92.2%-99.1%) vs. 80.9% (75.9%-86.1%)]. The molecular and EPclin scores were significant predictors of DRFR after adjusting for clinical variables, regardless of nodal status. Similar results were observed for late recurrence (5-15 years; HR, 4.52; 95% CI, 2.65-7.72; P < 0.0001). The EPclin score significantly added prognostic information to a late metastasis nomogram (CTS5 score; P < 0.001). CONCLUSIONS: This study demonstrates that EPclin can identify patients at low risk for early or late recurrence who may safely forgo adjuvant chemotherapy or extended endocrine therapy, respectively, regardless of nodal status.

Continued Endocrine Therapy Is Associated with Improved Survival in Patients with Breast Cancer Brain Metastases.

PURPOSE: Brain metastases (BMs) are a rare but devastating condition in estrogen receptor (ER)-positive metastatic breast cancer (MBC). Although endocrine therapy (ET) is the mainstay of treatment in this disease subtype, only case reports have been published concerning the activity of ET in BMs henceforth. Therefore, we aimed to systematically investigate the impact of ET after diagnosis of BM on outcome and clinical course of disease in patients with ER-positive MBC. EXPERIMENTAL DESIGN: Patient characteristics, detailed information about BMs including diagnosis-specific graded prognostic assessment class (DS-GPA), and clinical outcome were obtained by retrospective chart review for all patients treated for ER-positive breast cancer BMs between 1990 and 2017 at an academic care center. Overall survival (OS) was measured as the interval from diagnosis of BM until death or last date of follow-up. RESULTS: Overall, 198 patients [female: 195/198 (98.5%); male: 3/198 (1.5%)] with ER-positive breast cancer BMs were available for this analysis. Eighty-eight of 198 patients (44.4%) received ET after diagnosis of BM including aromatase inhibitors (AIs; letrozole, anastrozole, exemestane), tamoxifen, and fulvestrant. Median OS was significantly longer in patients receiving ET after diagnosis of BM compared with patients who did not (15 vs. 4 months, P < 0.001; log-rank test). No significant difference in terms of OS was observed between patients receiving AIs, tamoxifen, or fulvestrant. In patients with concomitant leptomeningeal carcinomatosis (LC), ET prolonged median OS significantly as well (7 vs. 3 months, P = 0.012; log-rank test). In a multivariate analysis including DS-GPA and ET, only treatment with ET after diagnosis of BM (HR, 0.69; 95% confidence interval, 0.48-0.99; P = 0.046) was associated with prognosis (Cox regression model). CONCLUSIONS: Continuing ET after BM diagnosis was associated with a significantly prolonged OS in this large single-center cohort. No substantial differences between substances were observed. These findings should be validated in a prospective cohort.

PIK3CA Mutational Status Is Associated with High Glycolytic Activity in ER+/HER2- Early Invasive Breast Cancer: a Molecular Imaging Study Using [18F]FDG PET/CT.

PURPOSE: In PIK3CA mutant breast cancer, downstream hyperactivation of the PI3K/AKT/mTOR pathway may be associated with increased glycolysis of cancer cells. The purpose of this study was to investigate the functional association of PIK3CA mutational status and tumor glycolysis in invasive ER+/HER2- early breast cancer. PROCEDURES: This institutional review board-approved retrospective study included a dataset of 67 ER+/HER2- early breast cancer patients. All patients underwent 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/X-ray computed tomography ([18F]FDG PET/CT) and clinico-pathologic assessments as part of a prospective study. For this retrospective analysis, pyrosequencing was used to detect PIK3CA mutations of exons 4, 7, 9, and 20. Tumor glucose metabolism was assessed semi-quantitatively with [18F]FDG PET/CT using maximum standardized uptake values (SUVmax). SUVmax values were corrected for the partial volume effect, and metabolic tumor volume was calculated using the volume of interest automated lesion growing function 2D tumor size, i.e., maximum tumor diameter was assessed on concurrent pre-treatment contrast-enhanced magnetic resonance imaging. RESULTS: PIK3CA mutations were present in 45 % of all tumors. Mutations were associated with a small tumor diameter (p < 0.01) and with low nuclear grade (p = 0.04). Glycolytic activity was positively associated with nuclear grade (p = 0.01), proliferation (p = 0.002), regional lymph node metastasis (p = 0.015), and metabolic tumor volume (p = 0.001) but not with tumor size/T-stage. In invasive ductal carcinomas, median SUVmax was increased in PIK3CA-mutated compared to wild-type tumors; however, this increase did not reach statistical significance (p = 0.05). Multivariate analysis of invasive ductal carcinomas revealed [18F]FDG uptake to be independently associated with PIK3CA status (p = 0.002) and nuclear tumor grade (p = 0.046). Size, volume, and regional nodal status had no influence on glycolytic activity. PIK3CA mutational status did not influence glycolytic metabolism in lobular carcinomas. Glycolytic activity and PIK3CA mutational status had no significant influence on recurrence-free survival or disease-specific survival. CONCLUSIONS: In ER+/HER2- invasive ductal carcinomas of the breast, glucose uptake is independently associated with PIK3CA mutations. Initial data suggest that [18F]FDG uptake reflects complex genomic alterations and may have the potential to be used as candidate biomarker for monitoring therapeutic response and resistance mechanisms in emerging therapies that target the PI3K/AKT/mTOR pathway.

Differential Claudin 3 and EGFR Expression Predicts BRCA1 Mutation in Triple-Negative Breast Cancer.

BRCA-1 mutation-associated triple-negative breast cancer (TNBC) has been hypothesized to exhibit a phenotype that is distinct from non-mutation carriers. We have analyzed immunohistochemically detected cytokeratins 5 and 14, epidermal growth factor receptor (EGFR), claudin (CLDN) 3, 4, and 7, and E-cadherin in 57 TNBC (32 BRCA1 and 8 BRCA2 tumors, 17 WT tumors). Positive staining of CLDN3 and negative EGFR expression in TNBC are associated with a BRCA1 mutation. EGFR and CLDN3 expression was able to predict the presence of BRCA1 mutation (area under curve 0.802, p < 0.001). This could help in guiding the decision for BRCA testing.

The breast lesion excision system (BLES) under stereotactic guidance cannot be used as a therapeutic tool in the excision of small areas of microcalcifications in the breast.

OBJECTIVE: The breast lesion excision system (BLES) is a new, automatic percutaneous breast biopsy device that excises single large specimens using radiofrequency cutting. The aim of this study was to determine whether BLES, under stereotactic guidance, can be used as a therapeutic tool in the assessment of small areas of microcalcifications in the breast by providing samples with clear margins. MATERIAL AND METHODS: In this retrospective study, 149 patients with suspicious (BIRADS 4 or 5) small areas of microcalcifications underwent stereotactic-guided BLES. Of these, 34 patients (22.8%) with microcalcifications that had a diameter smaller than the basket size (≤15mm) underwent both BLES and subsequent surgery. Histopathology findings from BLES and subsequent surgery were compared. Identical, underestimation and total excision findings were assessed. RESULTS: BLES revealed fourteen (41.1%) high-risk lesions, ten (29.4%) ductal carcinomas in situ, and ten (29.4%) invasive cancers. Identical results between BLES and surgery were seen in 17/34 (50%) lesions. Surgery confirmed total excision of BLES in 15/34 (44.1%) lesions. Underestimation was seen in 2/34 (5.8%) lesions. CONCLUSION: BLES allows accurate diagnosis of small areas of microcalcifications, with few underestimates. BLES is a diagnostic, but cannot be considered to be a therapeutic tool in the case of suspicious microcalcifications because total excision was seen in only 44.1% of these lesions. Studies are needed to address the therapeutic benefit of this procedure in solid lesions.

A simple classification system (the Tree flowchart) for breast MRI can reduce the number of unnecessary biopsies in MRI-only lesions.

OBJECTIVES: To assess whether using the Tree flowchart obviates unnecessary magnetic resonance imaging (MRI)-guided biopsies in breast lesions only visible on MRI. METHODS: This retrospective IRB-approved study evaluated consecutive suspicious (BI-RADS 4) breast lesions only visible on MRI that were referred to our institution for MRI-guided biopsy. All lesions were evaluated according to the Tree flowchart for breast MRI by experienced readers. The Tree flowchart is a decision rule that assigns levels of suspicion to specific combinations of diagnostic criteria. Receiver operating characteristic (ROC) curve analysis was used to evaluate diagnostic accuracy. To assess reproducibility by kappa statistics, a second reader rated a subset of 82 patients. RESULTS: There were 454 patients with 469 histopathologically verified lesions included (98 malignant, 371 benign lesions). The area under the curve (AUC) of the Tree flowchart was 0.873 (95% CI: 0.839-0.901). The inter-reader agreement was almost perfect (kappa: 0.944; 95% CI 0.889-0.998). ROC analysis revealed exclusively benign lesions if the Tree node was ≤2, potentially avoiding unnecessary biopsies in 103 cases (27.8%). CONCLUSIONS: Using the Tree flowchart in breast lesions only visible on MRI, more than 25% of biopsies could be avoided without missing any breast cancer. KEY POINTS: • The Tree flowchart may obviate >25% of unnecessary MRI-guided breast biopsies. • This decrease in MRI-guided biopsies does not cause any false-negative cases. • The Tree flowchart predicts 30.6% of malignancies with >98% specificity. • The Tree's high specificity aids in decision-making after benign biopsy results.

Pathological Complete Response to Neoadjuvant Trastuzumab Is Dependent on HER2/CEP17 Ratio in HER2-Amplified Early Breast Cancer.

Purpose: To evaluate whether pathologic complete response (pCR) to neoadjuvant trastuzumab is dependent on the level of HER2 amplification.Experimental Design: 114 HER2-overexpressing early breast cancer patients who had received neoadjuvant trastuzumab were included in this study. Absolute HER2 and chromosome 17 centromere (CEP17) were measured by in situ hybridization analysis, and associations were examined between HER2/CEP17 ratio and tumor pCR status (commonly defined by ypT0 ypN0, ypT0/is ypN0, and ypT0/is).Results: In trastuzumab-treated patients, ypT0 ypN0 was achieved in 69.0% of patients with high-level amplification (HER2/CEP17 ratio > 6), but only in 30.4% of tumors with low-level amplification (ratio ≤ 6; P = 0.001). When pCR was defined by ypT0/is ypN0 or ypTis, 75.9% and 82.8% of tumors with high-level amplification had a complete response, whereas only 39.1%, and 38.3% with low-level amplification achieved pCR (P = 0.002 and P < 0.001, respectively). Logistic regression revealed that tumors with high-level amplification had a significantly higher probability achieving ypT0 ypN0 (OR, 5.08; 95% confidence interval, 1.86-13.90; P = 0.002) than tumors with low-level amplification, whereas no other clinicopathologic parameters were predictive of pCR. The association between high-level HER2 amplification and pCR was almost exclusively confined to hormone receptor (HR)-positive tumors (ypT0 ypN0: 62.5% vs. 24.0%, P = 0.014; ypT0/is ypN0: 75.0% vs. 28.0%, P = 0.005; and ypT0/is: 87.5% vs. 28.0%, P < 0.001), and was largely absent in HR-negative tumors.Conclusions: An HER2/CEP17 ratio of >6 in the pretherapeutic tumor biopsy is associated with a significantly higher pCR rate, particularly in HER2/HR copositive tumors, and can be used as a biomarker to predict response before neoadjuvant trastuzumab is initiated. Clin Cancer Res; 23(14); 3676-83. ©2017 AACR.

Diffusion-weighted MRI of breast lesions: a prospective clinical investigation of the quantitative imaging biomarker characteristics of reproducibility, repeatability, and diagnostic accuracy.

Diffusion-weighted MRI (DWI) provides insights into tissue microstructure by visualization and quantification of water diffusivity. Quantitative evaluation of the apparent diffusion coefficient (ADC) obtained from DWI has been proven helpful for differentiating between malignant and benign breast lesions, for cancer subtyping in breast cancer patients, and for prediction of response to neoadjuvant chemotherapy. However, to further establish DWI of breast lesions it is important to evaluate the quantitative imaging biomarker (QIB) characteristics of reproducibility, repeatability, and diagnostic accuracy. In this intra-individual prospective clinical study 40 consecutive patients with suspicious findings, scheduled for biopsy, underwent an identical 3T breast MRI protocol of the breast on two consecutive days (>24 h). Mean ADC of target lesions was assessed (two independent readers) in four separate sessions. Reproducibility, repeatability, and diagnostic accuracy between examinations (E1, E2), readers (R1, R2), and measurements (M1, M2) were assessed with intraclass correlation coefficients (ICCs), coefficients of variation (CVs), Bland-Altman plots, and receiver operating characteristic (ROC) analysis with calculation of the area under the ROC curve (AUC). The standard of reference was either histopathology (n = 38) or imaging follow-up of up to 24 months (n = 2). Eighty breast MRI examinations (median E1-E2, 2 ± 1.7 days, 95% confidence interval (CI) 1-2 days, range 1-11 days) in 40 patients (mean age 56, standard deviation (SD) ±14) were evaluated. In 55 target lesions (mean size 25.2 ± 20.8 (SD) mm, range 6-106 mm), mean ADC values were significantly (P < 0.0001) higher in benign (1.38, 95% CI 1.27-1.49 × 10(-3)  mm(2) /s) compared with malignant (0.86, 95% CI 0.81-0.91 × 10(-) (3)  mm(2) /s) lesions. Reproducibility and repeatability showed high agreement for repeated examinations, readers, and measurements (all ICCs >0.9, CVs 3.2-8%), indicating little variation. Bland-Altman plots demonstrated no systematic differences, and diagnostic accuracy was not significantly different in the two repeated examinations (all ROC curves >0.91, P > 0.05). High reproducibility, repeatability, and diagnostic accuracy of DWI provide reliable characteristics for its use as a potential QIB, to further improve breast lesion detection, characterization, and treatment monitoring of breast lesions.