Angelo Di Leo

Prognostic and Predictive Value of Tumor-Infiltrating Lymphocytes in a Phase III Randomized Adjuvant Breast Cancer Trial in Node-Positive Breast Cancer Comparing the Addition of Docetaxel to Doxorubicin With Doxorubicin-Based Chemotherapy: BIG 02-98

PURPOSE Previous preclinical and clinical data suggest that the immune system influences prognosis and response to chemotherapy (CT); however, clinical relevance has yet to be established in breast cancer (BC). We hypothesized that increased lymphocytic infiltration would be associated with good prognosis and benefit from immunogenic CT-in this case, anthracycline-only CT-in selected BC subtypes. PATIENTS AND METHODS We investigated the relationship between quantity and location of lymphocytic infiltrate at diagnosis with clinical outcome in 2009 node-positive BC samples from the BIG 02-98 adjuvant phase III trial comparing anthracycline-only CT (doxorubicin followed by cyclophosphamide, methotrexate, and fluorouracil [CMF] or doxorubicin plus cyclophosphamide followed by CMF) versus CT combining doxorubicin and docetaxel (doxorubicin plus docetaxel followed by CMF or doxorubicin followed by docetaxel followed by CMF). Readings were independently performed by two pathologists. Disease-free survival (DFS), overall survival (OS), and interaction with type of CT associations were studied. Median follow-up was 8 years. RESULTS There was no significant prognostic association in the global nor estrogen receptor (ER) -positive/human epidermal growth factor receptor 2 (HER2) -negative population. However, each 10% increase in intratumoral and stromal lymphocytic infiltrations was associated with 17% and 15% reduced risk of relapse (adjusted P = .1 and P = .025), respectively, and 27% and 17% reduced risk of death in ER-negative/HER2-negative BC regardless of CT type (adjusted P = .035 and P = .023), respectively. In HER2-positive BC, there was a significant interaction between increasing stromal lymphocytic infiltration (10% increments) and benefit with anthracycline-only CT (DFS, interaction P = .042; OS, P = .018). CONCLUSION In node-positive, ER-negative/HER2-negative BC, increasing lymphocytic infiltration was associated with excellent prognosis. Further validation of the clinical utility of tumor-infiltrating lymphocytes in this context is warranted. Our data also support the evaluation of immunotherapeutic approaches in selected BC subtypes.

Results of the CONFIRM Phase III Trial Comparing Fulvestrant 250 mg With Fulvestrant 500 mg in Postmenopausal Women With Estrogen Receptor–Positive Advanced Breast Cancer

PURPOSE We compared fulvestrant 500 mg regimen with the approved dose of fulvestrant 250 mg per month for treatment of postmenopausal women with estrogen receptor-positive advanced breast cancer who experienced progression after prior endocrine therapy. PATIENTS AND METHODS Comparison of Faslodex in Recurrent or Metastatic Breast Cancer (CONFIRM) is a double-blind, parallel-group, multicenter, phase III study. Patients were randomly assigned to fulvestrant 500 mg (500 mg intramuscularly [IM] on day 0, then 500 mg IM on days 14 and 28 and every 28 days thereafter) or 250 mg every 28 days. Primary end point was progression-free survival (PFS). Secondary end points included objective response rate, clinical benefit rate (CBR), duration of clinical benefit (DoCB), overall survival (OS), and quality of life (QOL). RESULTS PFS was significantly longer for fulvestrant 500 mg (n = 362) than 250 mg (n = 374) (hazard ratio [HR] = 0.80; 95% CI, 0.68 to 0.94; P = .006), corresponding to a 20% reduction in risk of progression. Objective response rate was similar for fulvestrant 500 mg and 250 mg (9.1% v 10.2%, respectively). CBR was 45.6% for fulvestrant 500 mg and 39.6% for fulvestrant 250 mg. DoCB and OS were 16.6 and 25.1 months, respectively, for the 500-mg group, whereas DoCB and OS were 13.9 and 22.8 months, respectively, in the 250-mg group. Fulvestrant 500 mg was well tolerated with no dose-dependent adverse events. QOL was similar for both arms. CONCLUSION Fulvestrant 500 mg was associated with a statistically significant increase in PFS and not associated with increased toxicity, corresponding to a clinically meaningful improvement in benefit versus risk compared with fulvestrant 250 mg.

Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.

Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.

Dissecting the Heterogeneity of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) accounts for 15% to 20% of breast cancers. It is a heterogeneous disease, not only on the molecular level, but also on the pathologic and clinical levels. TNBC is associated with a significantly higher probability of relapse and poorer overall survival in the first few years after diagnosis when compared with other breast cancer subtypes. This is observed despite its usual high sensitivity to chemotherapy. In the advanced setting, responses observed with chemotherapy lack durability. Early-stage clinical studies suggested impressive potential when a poly (ADP-ribose) polymerase (PARP) inhibitor is given for the treatment of advanced TNBC with BRCA gene dysfunction. The molecular complexity of TNBC has led to proposed subclassifications, which will be of great value for the development of targeted therapies. In this review, we discuss the biology of TNBC at the pathologic and the molecular levels. We also elaborate on the role of systemic therapies and the results of the first phase III clinical trial evaluating the addition of iniparib, a novel investigational anticancer agent that does not possess characteristics typical of the PARP inhibitor class, in combination with chemotherapy in advanced TNBC.

Plasma microRNA 210 levels correlate with sensitivity to trastuzumab and tumor presence in breast cancer patients

Trastuzumab is part of the standard treatment for patients with human epidermal growth factor receptor 2 (HER‐2)‐positive breast cancer, but not all patients respond to trastuzumab. Altered microRNA (miR) expression levels in cancer cells have been correlated with prognosis and response to chemotherapy. The authors of this report hypothesized that altered miR expression levels in plasma are associated with sensitivity to trastuzumab in patients with HER‐2 positive breast cancer.

Phase III Trial Comparing Two Dose Levels of Epirubicin Combined With Cyclophosphamide With Cyclophosphamide, Methotrexate, and Fluorouracil in Node-Positive Breast Cancer

PURPOSE To compare a full-dose epirubicin-cyclophosphamide (HEC) regimen with classical cyclophosphamide, methotrexate, and fluorouracil (CMF) therapy and with a moderate-dose epirubicin-cyclophosphamide regimen (EC) in the adjuvant therapy of node-positive breast cancer. PATIENTS AND METHODS Node-positive breast cancer patients who were aged 70 years or younger were randomly allocated to one of the following treatments: CMF for six cycles (oral cyclophosphamide); EC for eight cycles (epirubicin 60 mg/m(2), cyclophosphamide 500 mg/m(2); day 1 every 3 weeks); and HEC for eight cycles (epirubicin 100 mg/m(2), cyclophosphamide 830 mg/m(2); day 1 every 3 weeks). RESULTS Two hundred fifty-five, 267, and 255 eligible patients were treated with CMF, EC, and HEC, respectively. Patient characteristics were well balanced among the three arms. One and three cases of congestive heart failure were reported in the EC and HEC arms, respectively. Three cases of acute myeloid leukemia were reported in the HEC arm. After 4 years of median follow-up, no statistically significant differences were observed between HEC and CMF (event-free survival [EFS]: hazards ratio [HR] = 0.96, 95% confidence interval [CI], 0.70 to 1.31, P =.80; distant-EFS: HR = 0.97, 95% CI, 0.70 to 1.34, P =.87; overall survival [OS]: HR = 0.97, 95% CI, 0.65 to 1.44, P =.87). HEC is more effective than EC (EFS: HR = 0.73, 95% CI, 0.54 to 0.99, P =.04; distant-EFS: HR = 0.75, 95% CI, 0.55 to 1.02, P =.06; OS HR = 0.69, 95% CI, 0.47 to 1.00, P =.05). CONCLUSION This three-arm study does not show an advantage in favor of an adequately dosed epirubicin-based regimen over classical CMF in the adjuvant therapy of node-positive pre- and postmenopausal women with breast cancer. Moreover, this study confirms that there is a dose-response curve for epirubicin in breast cancer adjuvant therapy.

HER-2 Gene Amplification, HER-2 and Epidermal Growth Factor Receptor mRNA and Protein Expression, and Lapatinib Efficacy in Women with Metastatic Breast Cancer

Purpose: Biomarkers from two randomized phase III trials were analyzed to optimize selection of patients for lapatinib therapy. Experimental Design: In available breast cancer tissue from EGF30001 (paclitaxel ± lapatinib in HER-2-negative/unknown metastatic breast cancer, n = 579) and EGF100151 (capecitabine ± lapatinib in HER-2-positive metastatic breast cancer, n = 399), HER-2 gene amplification by fluorescence in situ hybridization (FISH), HER-2 mRNA by reverse transcription-PCR (RT-PCR), HER-2 protein expression by HercepTest immunohistochemistry (IHC), epidermal growth factor receptor (EGFR) mRNA level by RT-PCR, and EGFR protein by IHC were analyzed and compared with clinical outcome. HER-2 was determined by FISH in an academic reference/research laboratory and in a large, high-volume commercial reference laboratory. Results: The HER-2 gene was amplified in 47% (344 of 733) and IHC was 3+ in 35% (279 of 798), with significant correlation (P < 0.01) between FISH and IHC. Positive EGFR immunostaining (IHC 1+, 2+, or 3+) in 28% (213 of 761) correlated with EGFR mRNA levels by RT-PCR (r = 0.59; P < 0.01). HER-2 gene amplification/overexpression was associated with improved clinical outcomes (progression-free survival; P < 0.001) in both trials. A significant improvement in outcome was seen in FISH-positive and IHC 0, 1+, or 2+ patients. HER-2 mRNA expression correlated with HER-2 FISH (r = 0.83) and IHC status (r = 0.72; n = 138). No correlation was found between EGFR expression (IHC or mRNA) and responsiveness to lapatinib regardless of HER-2 status. Although a significant correlation with lapatinib responsiveness was observed among “HER-2-negative” breast cancer patients in the large, high-volume commercial reference laboratory, this was not confirmed in the academic reference/research laboratory. Conclusions: Women with HER-2-positive metastatic breast cancer benefit from lapatinib, whereas women with HER-2-negative metastatic breast cancer derive no incremental benefit from lapatinib.

Adjuvant Chemotherapy With Sequential or Concurrent Anthracycline and Docetaxel: Breast International Group 02–98 Randomized Trial

BACKGROUND Docetaxel is more effective than doxorubicin for patients with advanced breast cancer. The Breast International Group 02-98 randomized trial tested the effect of incorporating docetaxel into anthracycline-based adjuvant chemotherapy and compared sequential vs concurrent administration of doxorubicin and docetaxel. METHODS Patients with lymph node-positive breast cancer (n = 2887) were randomly assigned to one of four treatments: 1) sequential control (four cycles of doxorubicin at 75 mg/m2, followed by three cycles of cyclophosphamide, methotrexate, and 5-fluorouracil [CMF]); 2) concurrent control (four cycles of doxorubicin at 60 mg/m2 plus cyclophosphamide at 600 mg/m2, followed by three cycles of CMF); 3) sequential docetaxel (three cycles of doxorubicin at 75 mg/m2, followed by three cycles of docetaxel at 100 mg/m2, followed by three cycles of CMF); 4) concurrent docetaxel (four cycles of doxorubicin at 50 mg/m2 plus docetaxel at 75 mg/m2, followed by three cycles of CMF). The primary comparison evaluated the efficacy of including docetaxel regardless of schedule and was planned after 1215 disease-free survival (DFS) events (ie, relapse, second primary cancer, or death from any cause). Docetaxel and control treatment groups were compared by log-rank tests, and hazard ratios (HR) of DFS events were calculated by Cox modeling. All statistical tests were two-sided. RESULTS Due to a lower-than-anticipated rate of relapse, this analysis was performed after 5 years with 732 events. Patients in control arms had a 5-year DFS of 73% (95% confidence interval [CI] = 70% to 75%). Docetaxel treatment resulted in an improvement in DFS of borderline statistical significance compared with control treatment (HR = 0.86, 95% CI = 0.74 to 1.00; P = .05). However, DFS in the sequential docetaxel arm was better than that in the concurrent docetaxel arm (HR = 0.83, 95% CI = 0.69 to 1.00) and in the sequential control arm (HR = 0.79, 95% CI = 0.64 to 0.98). CONCLUSIONS Incorporating docetaxel into anthracycline-based therapy resulted in an improvement in DFS that was of borderline statistical significance. However, important differences may be related to doxorubicin and docetaxel scheduling, with sequential but not concurrent administration, appearing to produce better DFS than anthracycline-based chemotherapy.

Multifactorial Approach to Predicting Resistance to Anthracyclines

PURPOSE Validated biomarkers predictive of response/resistance to anthracyclines in breast cancer are currently lacking. The neoadjuvant Trial of Principle (TOP) study, in which patients with estrogen receptor (ER) -negative tumors were treated with anthracycline (epirubicin) monotherapy, was specifically designed to evaluate the predictive value of topoisomerase II-α (TOP2A) and develop a gene expression signature to identify those patients who do not benefit from anthracyclines. PATIENTS AND METHODS The TOP trial included 149 patients, 139 of whom were evaluable for response prediction analyses. The primary end point was pathologic complete response (pCR). TOP2A and gene expression profiles were evaluated using pre-epirubicin biopsies. Gene expression data from ER-negative samples of the EORTC (European Organisation for Research and Treatment of Cancer) 10994/BIG (Breast International Group) 00-01 and MDACC (MD Anderson Cancer Center) 2003-0321 neoadjuvant trials were used for validation purposes. RESULTS A pCR was obtained in 14% of the evaluable patients in the TOP trial. TOP2A amplification, but not protein overexpression, was significantly associated with pCR (P ≤ .001 v P ≤ .33). We developed an anthracycline-based score (A-Score) combining three signatures: a TOP2A gene signature and two previously published signatures related to tumor invasion and immune response. The A-Score was characterized by a high negative predictive value ([NPV]; NPV, 0.98; 95% CI, 0.90 to 1.00) overall and in the human epidermal growth factor receptor 2 (HER2) -negative and HER2-positive subpopulations. Its performance was independently confirmed in the anthracycline-based arms of the two validation trials (BIG 00-01: NPV, 0.83; 95% CI, 0.64 to 0.94 and MDACC 2003-0321: NPV, 1.00; 95% CI, 0.80 to 1.00). CONCLUSION Given its high NPV, the A-Score could become, if further validated, a useful clinical tool to identify those patients who do not benefit from anthracyclines and could therefore be spared the non-negligible adverse effects.

Management of triple negative breast cancer

Triple negative breast cancer (TNBC) accounts for approximately 15% of breast cancer cases. TNBC is an immunohistochemically defined subtype, with significant diversity within the subtype. Generally TNBC occurs in younger women and is marked by high rates of relapse, visceral and CNS metastases, and early death. Current therapy fails to curtail the innate aggressive behaviour of TNBC in the majority of patients. The poor prognosis coupled with a lack of targeted use of therapies is reflected in the high mortality. In a minority of patients with highly chemosensitive disease, no robust clinical evidence exists to guide use of current cytotoxics. Critical to optimal future management are accurate identification of truly triple negative disease and adequately powered prospective TNBC trials to establish treatment efficacy and define predictive biomarkers.