Prognostic and Predictive Value of Tumor-Infiltrating Lymphocytes in Breast Cancer

Recent attention has focused on the antitumor immune response in breast cancer, in particular, the role of tumor-infiltrating lymphocytes (TILs). In certain breast cancer subtypes, the presence of TILs has prognostic value and is associated with clinical outcome and improved survival. The potential predictive value of TILs has also been investigated in regard to cytotoxic and antihuman epidermal growth factor receptor 2 (HER-2) therapies, but could also serve as a selection marker for novel immunotherapies. In this article, we review the current literature on TILs in breast cancer and discuss how TILs are emerging as a promising biomarker in mediating antitumor immunity.     

Prognostic Value of Pretreatment Neutrophil-to-Lymphocyte Ratio in HER2-Positive Metastatic Breast Cancer

This study aimed to examine the prognostic value of the neutrophil-to-lymphocyte ratio (NLR) and other clinicopathological features in HER2+ MBC patients who received first-line anti-HER2 therapy. A total of 129 patients were assigned to NLR-low and NLR-high groups based on a cutoff value of 3.0 at baseline. Peripheral blood lymphocyte subsets and gene mutations in circulating tumor DNA were analyzed by flow cytometry and Next-generation sequencing, respectively. Survival was evaluated by the Kaplan–Meier method and Cox regression analysis. Of the 129 patients, 77 and 52 were assigned to the NLR-low (≤3) and NLR-high (>3) groups, respectively. Compared with NLR-high patients, the NLR-low patients had significantly longer median progression-free survival (PFS) (11.7 vs. 7.7 months) (p = 0.001, HR = 2.703 95% CI 1.543–4.736 and overall survival (OS) (37.4 vs. 28.7 months) (p = 0.044, HR = 2.254 95% CI 1.024–4.924). Furthermore, this association was independent of metastatic sites or estrogen receptor status. Peripheral blood CD3+ (p = 0.034) and CD4+ (p = 0.010) T cell numbers were significantly higher in the NLR-low group than the NLR-high group. The mutational profile of MBC was generally similar between the two groups. Baseline NLR was a prognostic factor of PFS and OS for patients with HER2+ MBC in the first-line setting. These results may facilitate the selection of patients who will benefit most from anti-HER2 treatment.     

Prognostic Value of Natural Killer Cells Besides Tumor-Infiltrating Lymphocytes in Breast Cancer Tissues

BackgroundEach subgroup of immune cells has a different prognostic role in breast cancer; however, the prognostic impact of tumor-infiltrating natural killer cells (TINKs) is still not well established. Our aim was to assess the prognostic impact of natural killer (NK) cells in breast carcinomas.Materials and MethodsNK cells infiltration were assessed by immunohistochemistry (IHC). Statistical analyses were performed to evaluate the correlation of NK cells with clinical-pathological features and outcome.ResultsCD56 IHC was realized in 126 patients. NK cells infiltration showed significant and positive association with tumor high Scarff-Bloom-Richardson (SBR) grade. NK cells were significantly associated with HER2-positive breast cancer and triple-negative breast cancer subtypes. Analyses showed significant and inverse correlation with progesterone and estrogen receptors expression status. High NK cells were significantly related to high Ki-67 labeling index. Our data showed that high NK cells infiltrate was significantly associated with tumor-infiltrating lymphocytes in breast cancer tissues.At a median follow-up of 5.5 years, high CD56 expression (≥ 5 cells/10 high power field) was associated significantly with a good overall survival and with good disease-free survival.ConclusionIn this study, we assessed the important prognostic role of TINKs in breast carcinomas, which seems to be evident despite its association with aggressive pathological features. Thus evaluation of NK cells can be standardized and integrated in daily routine.     

Predictive and Prognostic Significance of p27, Akt,PTEN and PI3K Expression in HER2-Positive Metastatic Breast Cancer

Background: The phosphatidylinositol 3’-kinase/Akt (PI3K/Akt) pathway is a key regulator for HER2-overexpressing breast cancer, but data about whether activation of PI3K/Akt is associated with poor prognosisand resistance to trastuzumab therapy is controversial. In this study we investigated predictive and prognosticsignificance of expression of p27, Akt, PTEN and PI3K, which are components of the PI3K/Akt signaling pathway,in HER2-positive metastatic breast cancer (MBC), retrospectively. Materials and Methods: Fifty-four HER2-positive MBC patients who had received first-line trastuzumab-based therapy were recruited for the study group.All of the patient’s breast tissue samples were examined for p27 and Akt expression. In addition, twenty-fivepatients with sufficient amount of tumor tissue were also examined for PTEN and PI3K expression. p27, Akt,PTEN and PI3K were evaluated by immunohistochemistry and their relationship with patient demographicfeatures, tumor characteristics, response to trastuzumab-based treatment and survival outcomes were analyzed.Results: p27, Akt, PTEN and PI3K were positive in 25.9%, 70.4%, 24% and 96% of the cases, respectively.Nomne were significantly associated with response to trastuzumab and time to progression (TTP). A trend towardstatistical significance for longer overall survival (OS) was found for PTEN-positive patients (p=0.058); there wasno significant relationship between the other immunohistochemical variables and OS. When we analyzed groupsregarding co-expression, the PTEN-negative/Akt-negative group had a significantly lower objective responserate (ORR) (20% vs 80%, p=0.023) and the PTEN-negative/p27-negative and PTEN-negative/Akt-negativegroups had significantly lower median OS compared to other patients (26.4 months vs 76.1 months, p=0.005 and25.6 months vs 52.0 months, p=0.007, respectively). Conclusions: p27, Akt, PTEN and PI3K expression is notstatistically significantly associated with ORR, TTP and OS, individually. However, the combined evaluationof p27, Akt and PTEN could be helpful to predict the response to trastuzumab-based therapy and prognosis inHER2-positive MBC.     

Prognostic Value of Tumor Infiltrating Lymphocytes in Locally Advanced HER2 Enriched Breast Cancer

Purpose: We aim to study the association between stromal tumor infiltrating lymphocytes (TILs) level and disease free survival (DFS) in a group of ER and PR negative, HER2+ locally advanced breast cancer patients who underwent curative intent surgery. Methods: This is a retrospective cohort study including 66 locally advanced hormone receptor-negative; HER2+ breast cancer patients presented between 2013 and 2015 at NCI-Cairo, Egypt. Enrolled patients had at least clinically T3 and/or node positive disease either clinically or radiologically. Metastatic workup included CT and bone scans or PET-CT. Patients with hormone receptor positive, HER2 negative, inadequate paraffin block and who lost follow up before or immediately after curative surgery were excluded. Patients were followed from breast surgery till relapse date for a minimum of 36 months. TILs and CD8 antigen were assessed on paraffin-embedded blocks using immunohistochemistry. Results: Patients with a median age of 52 years presented with clinical T3 stage (53%) and N1 stage (61%). Modified radical mastectomy was performed in 79%. Only 41% received neoadjuvant chemotherapy and 56% received trastuzumab. TILs were 50, 17 and 33% for absent, intermediate and extensive groups and CD8+ lymphocytes were present in 80% of cases. At the end of follow-up period, 23 patients (35%) were found to have disease recurrence either loco-regional (22%) or distant (78%). TILs were 14, 4 and 5% for absent, intermediate and extensive respectively; while CD8+ lymphocytes were absent in 6% and present (≥1%) in 17%. Higher DFS was recorded for patients with extensive TILs level only who received trastuzumab. Conclusion: High TILs is good prognosis in HER2 enriched breast cancer provided that patients received HER2 directed therapy. Moreover, CD8+ lymphocytes are highly representative and maybe used as an alternative for TILs. We recommend considering TILs and specifically CD8+ as one of the risk factors that predict prognosis of HER2+ breast cancer.     

Tumor-Infiltrating Lymphocytes in Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer Receiving Neoadjuvant Docetaxel,Carboplatin, Trastuzumab,and Pertuzumab

PurposeThe tumor-infiltrating lymphocytes (TILs) expression in breast cancer is a positive prognostic marker for certain breast cancer subtypes. We evaluated the efficacy of dual anti-human epidermal growth factor receptor 2 (HER2) blockade in HER2-positive breast cancer and hypothesized that high TILs tumors are associated with better outcomes.MethodsA total of 176 patients who were treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP) between December 2015 and December 2018 were reviewed. They were grouped based on a cut-off value of the stromal TILs grade (≤ 20% TILs, > 20% TILs).ResultsIn total, 107 patients (60.8%) achieved pathological complete response (pCR). Hormone receptor (HR)-negativity (p = 0.001) and a high TILs grade (p = 0.022) were independent predictors of pCR. Among the HR-negative patients, high TILs tumors were significantly associated with pCR (p = 0.035).ConclusionHR status and the TILs grade are significantly correlated with pCR in dual anti-HER2 neoadjuvant therapy. The evaluation of the TILs at baseline may be beneficial for predicting pCR in HER2-positive breast cancer.     

Serial Serum HER2 Measurements for the Detection of Breast Cancer Recurrence in HER2-Positive Patients

Purpose

The measurement of serum human epidermal growth factor receptor 2 (HER2) extracellular domain levels is a well-established method for evaluating whether a metastatic HER2-positive breast cancer patient will respond to HER2-targeted treatment. However, little is known about the value of serum HER2 for detecting disease relapse following curative surgical treatment in breast cancer patients. The purpose of this study was to evaluate the sensitivity of serum HER2, carcinoembryonic antigen (CEA), and carcinoma antigen 15-3 (CA 15-3) for the detection of disease recurrence in postoperative breast cancer patients with a primary HER2-positive tumor.

Methods

Serial measurements were taken of serum HER2, CEA, and CA 15-3 levels in patients with primary invasive HER2-positive breast cancer who underwent curative surgical treatment between January 2008 and December 2010. Following treatment, serum HER2 levels were monitored every 6 months using a chemiluminescence immunoassay.

Results

Overall, 264 patients were analyzed in this retrospective study. The median follow-up period was 27.7 months, and 24 patients relapsed during follow-up. The sensitivity of serum HER2, CEA, and CA 15-3 for the detection of disease recurrence was 37.5%, 25.1%, and 12.5%, respectively. Sensitivity increased to 45.8% when all three tumor markers were combined in the analysis. In a subgroup of patients without liver disease, the sensitivity of serum HER2, CEA, and CA 15-3 was 57.1%, 21.4%, and 14.3%, respectively. Of the 264 patients in this study, 80 patients had chronic hepatitis, liver cirrhosis, or abnormal aspartate aminotransferase or alanine aminotransferase levels during the follow-up period. Following the exclusion of these patients, the sensitivity of serum HER2 for the detection of disease recurrence increased to 57.1%.

Conclusion

Serial serum HER2 measurement may be useful for the detection of disease relapse in patients with HER2-positive breast cancer. Abnormal liver function can result in elevated serum HER2 in the absence of disease recurrence.     

HER2 positive breast cancer patients having HER2 loss after neoadjuvant chemotherapy should still be treated with adjuvant anti-HER2 treatment

Breast Cancer Research and Treatment -     

Clinical Significance of PD-L1 Expression and CD8-Positive Tumor-Infiltrating Lymphocytes in Patients with Cavitary Lung Adenocarcinoma

Cavitary lung cancer is a rare type of lung cancer. Generally, the relationship between cavitary lung adenocarcinoma (LUAD) and specific immune checkpoints remains unknown. In this study, we aimed to detect the expression of programmed cell death ligand-1(PD-L1) and the density of CD8-positive (CD8⁺) tumor-infiltrating lymphocytes (TILs) to evaluate their clinicopathological significance in the case of patients with cavitary LUAD. This study included 65 patients with cavitary LUAD. Patient specimens were obtained from surgery. The expression of PD-L1 protein and CD8⁺ TIL status was detected by traditional immunohistochemistry and multiplex quantitative immunofluorescence technology. The correlation of PD-L1 expression and CD8⁺ TIL status was assessed along with clinicopathological parameters. This included evaluation of overall survival in cavitary LUAD patients based on the follow-up data. High expression of PD-L1 protein was detected in the cancerous tissues of cavitary LUAD patients, whose positive rate was recorded at 44.6% (29/65). PD-L1 expression level was significantly related with the lymph node (P = 0.001), TNM stage (P = 0.024), and CD8⁺ TIL status (rs = −0.272, P = 0.025). High PD-L1 expression indicated high mortality rate (P < 0.001); however, the CD8⁺ TIL group showed better survival in cavitary LUAD patients (P = 0.011). This phenotype, with high PD-L1 expression and low CD8⁺ TILs, can predict poorer overall survival of patients with cavitary LUAD compared with the other phenotypes. Moreover, CD8⁺ TIL level was an independent good prognosis factor. Initially, we reported that PD-L1 is upregulated in cavitary LUAD patients and that high expression of PD-L1 negatively correlates with CD8⁺ TIL status. High PD-L1 expression and low CD8⁺ TILs can predict decreased overall survival of patients with cavitary LUAD.     

Prognostic value of Bcl‐2 in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy

We have analyzed the predictive/prognostic value of Bcl‐2 protein in breast cancer patients treated with neoadjuvant chemotherapy. One hundred and ten patients were submitted to two different chemotherapeutic regimens: a) 5‐fluorouracil, adriamycin or epirubicin, and cyclophosphamide (FAC/FEC) during 2–6 cycles before surgery and 3 or 4 additional cycles of FAC/FEC after surgery (n=40) and b) doxorubicin (D) 75mg/m2 or epirubicin (E) 120mg/m2 during 4 cycles before surgery, and 6 cycles of cyclophosphamide, methotrexate, and 5‐fluorouracil (CMF) after surgery (n=70). Bcl‐2 expression, evaluated by immunohistochemistry, did not change significantly after chemotherapy and was not related to clinical/pathological response. In FAC/FEC group, Bcl‐2 positive expression after chemotherapy correlated with better disease free survival (DFS) and overall survival (OS) (P=0.008 and P=0.001). In D/E group, Bcl‐2 also correlated with better DFS and OS (P=0.03 and P=0.054) in the post‐chemotherapy biopsies. An unusual nuclear localization of Bax was observed in some biopsies, but this localization did not correlate with the tumor response or outcome of the patients. We found that a high Bcl‐2 expression had no predictive value but had prognostic value in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy.     

Hormone receptor status and pathologic response of HER2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab

Background: The aim of this study was to compare the extent of pathologic response in patients with HER2-positive (HER2+) breast cancer treated with standard neoadjuvant chemotherapy, with or without trastuzumab (H), according to hormone receptor (HR) status.Patients and methods: We included 199 patients with HER2+ breast cancer from three successive cohorts of neo-adjuvant chemotherapy on the basis of paclitaxel (Taxol) (P) administered weekly (w) or three weekly (3-w), followed by 5-fluorouracil (F), doxorubicin (A) or epirubicin (E), and cyclophosphamide (C). Residual cancer burden (RCB) was determined from pathologic review of the primary tumor and lymph nodes and was classified as pathologic complete response (pCR) or minimal (RCB-I), moderate (RCB-II), or extensive (RCB-III) residual disease.Results: In HR-positive (HR+) cancers, a higher rate of pathologic response (pCR/RCB-I) was observed with concurrent H + 3-wP/FEC (73%) than with 3-wP/FEC (34%, P = 0.002) or wP/FAC (47%; P = 0.02) chemotherapy alone. In HR-negative (HR-) cancers, there were no significant differences in the rate of pathologic response (pCR/RCB-I) from 3-wP/FAC (50%), wP/FAC (68%), or concurrent H + 3-wP/FEC (72%).Conclusions: Patients with HR+/HER2+ breast cancer obtained significant benefit from addition of trastuzumab to P/FEC chemotherapy; pathologic response rate was similar to that seen in HR-/HER2+ breast cancers.     

Prognostic and Predictive Relevance of Tumor-Infiltrating Lymphocytes in Squamous Cell Head–Neck Cancer Patients Treated with Radical Radiotherapy/Chemo-Radiotherapy

Microenvironmental conditions control the entrance and thriving of cytotoxic lymphocytes in tumors, allowing or preventing immune-mediated cancer cell death. We investigated the role of tumor-infiltrating lymphocyte (TIL) density in the outcome of radiotherapy in a series of squamous cell head–neck tumors (HNSCC). Moreover, we assessed the link between markers of hypoxia and TIL density. One-hundred twenty-one patients with HNSCC treated prospectively with radical radiotherapy/chemo-radiotherapy were analyzed. The assessment of TIL density was performed on hematoxylin and eosin biopsy sections before radiotherapy. TIL density ranged from 0.8 to 150 lymphocytes per ×40 optical field (median 27.5). Using the median value, patients were grouped into two categories of low and high TIL density. Early T-stage tumors had a significantly higher TIL density (p < 0.003), but we found no association with N-stage. Overexpression of HIF1α, HIF2α, and CA9 was significantly linked with poor infiltration by TILs (p < 0.03). A significant association of high TIL density with better disease-specific overall survival and improved locoregional relapse-free survival was noted (p = 0.008 and 0.02, respectively), which was also confirmed in multivariate analysis. It is concluded that HNSCC phenotypes that allow for the intratumoral accumulation of lymphocytes have a better outcome following radical radiotherapy/chemo-radiotherapy. Intratumoral-activated HIF- and CA9-related pathways characterize immunologically cold tumors and may be used as targets for therapeutic interventions.     

Optimal Strategies for Successful Initiation of Neratinib in Patients with HER2-Positive Breast Cancer

Neratinib is an irreversible, pan-human epidermal growth factor inhibitor that has shown efficacy across human epidermal growth factor receptor 2 (HER2)-positive breast cancer settings. Neratinib is indicated for use as extended adjuvant therapy for HER2-positive early-stage breast cancer or, in combination with capecitabine, in the treatment of HER2-positive metastatic breast cancer. The primary tolerability concern with neratinib is diarrhea, and severe diarrhea early in treatment can lead to a substantial proportion of patients discontinuing neratinib, which may lead to reduced or nonexistent efficacy. In order to establish a set of treatment recommendations for use of neratinib, on May 12, 2020, an expert panel of oncologists and gastroenterologists met virtually to discuss the role of neratinib in the treatment of patients with HER2-positive breast cancer. The panel reviewed the current data on neratinib, including efficacy across settings and diarrhea management strategies. Based on these data and their clinical experience, the panelists developed a set of recommendations to guide selection of patients for neratinib, implement weekly dose escalation at initiation of therapy, and prophylactically manage diarrhea.     

Developments in the Management of Metastatic HER2-Positive Breast Cancer: A Review

Approximately 20% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2), providing an actionable target for many different therapies. In the metastatic setting, prognosis has improved greatly with the use of anti-HER2 drugs such as trastuzumab, pertuzumab, and trastuzumab-emtansine. In the third line setting and beyond, several emerging treatments have shown benefits, including novel small molecule targeted agents and antibody-drug conjugates. Systemic treatment of brain metastases in HER2-positive patients and the role of endocrine-based treatment for patients with hormone receptor (HR) positive disease remain areas of research interest. This article will review the current approach to systemic management of metastatic HER2-positive breast cancer in Canada, and present novel treatments that may be available in the near future.     

Stromal lymphocyte infiltration after neoadjuvant chemotherapy is associated with aggressive residual disease and lower disease-free survival in HER2-positive breast cancer

BackgroundThe role of tumor-infiltrating lymphocytes (TILs) in breast cancer has been extensively studied over the last decade. High TILs levels have been associated with pathological response rate in the neoadjuvant setting and with better outcomes in the adjuvant setting. However, little attention has been paid to changes in TILs and residual TIL levels after neoadjuvant chemotherapy (NAC). We investigated TIL levels before, after chemotherapy, and their dynamics during treatment; and we assessed the correlation of these levels with response to NAC and prognosis.Materials and methodsWe identified 175 patients with primary HER2-positive breast cancers receiving NAC+/− trastuzumab between 2002 and 2011. Microbiopsy specimens and paired surgical samples were evaluated for stromal lymphocyte infiltration. Univariate and multivariate analyses were carried out to assess the association of clinical and pathological factors with pathological complete response (pCR) and disease-free survival.ResultsBaseline TIL levels were not significantly associated with pCR. TIL levels decreased during treatment in 78% of the patients. The magnitude of the decrease was strongly associated with pCR. After chemotherapy, TIL levels were high in tumors displaying aggressive patterns (high residual cancer burden score, mitotic index >22, tumor cellularity >5%). In the population with residual disease, TIL levels >25% at the end of NAC were significantly associated with an adverse outcome (TILs >25%, HR = 7.98, P = 0.009) after multivariate analyses including BMI, post-NAC mitotic index and tumor grade.ConclusionA decrease in TIL levels during chemotherapy was positively associated with response to treatment. In tumor failing to achieve pCR, post-NAC lymphocytic infiltration was associated with higher residual tumor burden and adverse clinical outcome. Further studies are required to characterize immune infiltration in residual disease to identify candidates who could benefit from second-line therapy trials including immune checkpoint inhibitors.     

HER2 and response to anthracycline-based neoadjuvant chemotherapy in breast cancer

BackgroundThe predictive role of human epidermal growth factor receptor 2 (HER2) to adjuvant anthracycline-based chemotherapy remains controversial. Here, we investigated the association between HER2 status and pathological response in breast cancer patients who received neoadjuvant anthracycline-based regimens.Patients and methodsWomen (n = 538) with operable primary breast cancer received neoadjuvant anthracycline-based chemotherapy. Pathological complete response (pCR) was defined as no invasive breast tumor cells in breast after completion of neoadjuvant chemotherapy. HER2 status was determined by immunohistochemistry and/or by fluorescence in situ hybridization in core biopsy breast cancer tissue obtained before initiation of neoadjuvant chemotherapy.ResultsIn this cohort of 538 patients, 23.9% of patients achieved a pCR in their breast. HER2-positive tumors had a lower rate of pCR than did HER2-negative tumors (14.7% versus 25.7%, P = 0.013); negative HER2 status remained as an independent favorable predictor of pCR after adjusted for age, estrogen receptor, progesterone receptor, tumor size, chemotherapy cycles, and tumor grade in a multivariate analysis (odds ratio = 3.14; 95% confidence interval = 1.60–6.16, P = 0.001). Furthermore, patients with a pCR had a higher 3-year disease-free survival (DFS) rate than did patients without a pCR (P = 0.007).ConclusionWomen with HER2-negative breast cancers rather than HER2-positive tumors benefit from anthracycline-based neoadjuvant chemotherapy.     

HER2-low status and response to neoadjuvant chemotherapy in HER2 negative early breast cancer

Purpose

Androgen receptor (AR) expression occurs in up to 86% of human epidermal growth factor receptor 2-positive (HER2+) breast cancers. In vitro, AR inhibitors enhance antitumor activity of trastuzumab, an anti-HER2 antibody, in trastuzumab-resistant HER2+?cell lines. This open-label, single-arm, phase II study evaluated the efficacy and safety of enzalutamide, an AR-signaling inhibitor, in patients with advanced HER2+?AR+?breast cancer previously treated with trastuzumab.

Methods

Eligible patients had measurable or non-measurable evaluable disease per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, Eastern Cooperative Oncology Group status?≤?1, no history of brain metastases, and previously received?≥?1 anti-HER2 regimen for advanced disease. Patients received 160 mg oral enzalutamide daily and 6 mg/kg intravenous trastuzumab every 21 days until disease progression or unacceptable toxicity. Primary end point was clinical benefit rate at 24 weeks (CBR24); secondary end points included progression-free survival (PFS) and safety.

Results

Overall, 103 women were enrolled [median age 60 years (range?34–83)]; 62% had received?≥?3 lines of prior anti-HER2 therapy. CBR24, comprising patients with confirmed partial responses (5%) and durable stable disease at 24 weeks (19%), was 24% in the efficacy evaluable set (n?=?89). CBR24 did not seem related to AR-expression levels or hormone receptor status. Median PFS was 3.4 months (95% confidence interval 2.0–3.8). Overall, 97 (94%) patients experienced treatment-emergent adverse events (TEAEs), with fatigue most common (34%). Dyspnea (4%) and malignant neoplasm progression (3%) were the only TEAEs grade?≥?3 reported in?≥?3 patients. 22 patients (21%) reported serious TEAEs. Four patients (4%) experienced fatal, non-drug-related TEAEs.

Conclusions

Enzalutamide plus trastuzumab was well tolerated, and a subset of patients in this heavily pretreated population had durable disease control. Determination of biomarkers is needed to identify patients most likely to benefit from this combination.

ClinicalTrials.gov number

NCT02091960