Juan Pablo Fusco

Tumor-produced interleukin-8 attracts human myeloid-derived suppressor cells and elicits extrusion of neutrophil extracellular traps (NETs)

Purpose: Myeloid-derived suppressor cells (MDSC) are considered an important T-cell immunosuppressive component in cancer-bearing hosts. The factors that attract these cells to the tumor microenvironment are poorly understood. IL8 (CXCL8) is a potent chemotactic factor for neutrophils and monocytes. Experimental Design: MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n = 10) and advanced cancer patients (n = 28). In chemotaxis assays, sorted granulocytic and monocytic MDSC were tested in response to recombinant IL8, IL8 derived from cancer cell lines, and patient sera. Neutrophil extracellular traps (NETs) formation was assessed by confocal microscopy, fluorimetry, and time-lapse fluorescence confocal microscopy on short-term MDSC cultures. Results: IL8 chemoattracts both granulocytic (GrMDSC) and monocytic (MoMDSC) human MDSC. Monocytic but not granulocytic MDSC exerted a suppressor activity on the proliferation of autologous T cells isolated from the circulation of cancer patients. IL8 did not modify the T-cell suppressor activity of human MDSC. However, IL8 induced the formation of NETs in the GrMDSC subset. Conclusions: IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control. Clin Cancer Res; 22(15); 3924–36. ©2016 AACR.

Strategies to design clinical studies to identify predictive biomarkers in cancer research

The discovery of reliable biomarkers to predict efficacy and toxicity of anticancer drugs remains one of the key challenges in cancer research. Despite its relevance, no efficient study designs to identify promising candidate biomarkers have been established. This has led to the proliferation of a myriad of exploratory studies using dissimilar strategies, most of which fail to identify any promising targets and are seldom validated. The lack of a proper methodology also determines that many anti-cancer drugs are developed below their potential, due to failure to identify predictive biomarkers. While some drugs will be systematically administered to many patients who will not benefit from them, leading to unnecessary toxicities and costs, others will never reach registration due to our inability to identify the specific patient population in which they are active. Despite these drawbacks, a limited number of outstanding predictive biomarkers have been successfully identified and validated, and have changed the standard practice of oncology. In this manuscript, a multidisciplinary panel reviews how those key biomarkers were identified and, based on those experiences, proposes a methodological framework-the DESIGN guidelines-to standardize the clinical design of biomarker identification studies and to develop future research in this pivotal field.

Clinical management of small-cell carcinoma of the urinary tract: a 10-year single-center's experience.

BACKGROUND Small-cell carcinoma (SCC) comprises 1% of primary bladder tumors and approximately 2% of prostate neoplasms. Metastatic disease at diagnosis is common, and survival outcomes are extremely poor. There is controversy about the ideal clinical management of these patients. The neuron-specific enolase (NSE) serum levels have never been studied in patients with small-cell carcinoma of the urinary tract (SCCUT). PATIENTS AND METHODS We report the clinical outcome of 12 consecutive SCCUT patients treated during the past 10 years. We also study the NSE levels at diagnosis and during treatment. RESULTS Patients with limited disease (LD) experienced a non-significant longer progression-free survival (PFS) and overall survival (OS) compared with extensive disease (ED) subjects. Patients with bladder SCC showed a significantly higher median PFS compared with prostate SCCUT patients (22 vs. 6 months; P = .034), although that difference did not impact on a significant longer OS. NSE levels decreased during chemotherapy administration in all patients with ED and baseline high levels. CONCLUSIONS Our patients showed a poor prognosis as described in previous studies. A better outcome for patients with bladder SCC compared with prostate SCC could be suggested. Serum NSE levels should be further evaluated to prove its potential use in early diagnosis and treatment monitoring during chemotherapy.

Epidermal Growth Factor Receptor targeting in non-small cell lung cancer: revisiting different strategies against the same target.

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the paradigm of treatment in non-small cell lung cancer (NSCLC). The molecular biology study of EGFR has led to clinical trials that select patients more accurately, regarding the presence of EGFR activating mutations. Nonetheless, a lack of response or a temporary condition of the response has been detected in patients on EGFR TKIs. This has urged to study potential resistance mechanisms underneath. The most important ones are the presence of secondary mutations in EGFR, such as T790M, or the overexpression of mesenchymal-epithelial transition factor (MET) that may explain why patients who initially respond to EGFR TKIs, may ultimately become refractory. Several approaches have been taken and new drugs both targeting EGFR resistance-mutation or MET are currently being developed. Here we review and update the EGFR biological pathway as well as the clinical data leading to approval of the EGFR TKIs currently in the market. New compounds under investigation targeting resistance mutations or dually targeting EGFR and other relevant receptors are also reviewed and discussed.

EGFR mutation testing in nonsmall cell lung cancer patients by using cytology specimens: when the tissue is no longer the issue.

We have read with great interest the article by Billah et al reporting their experiences with the molecular testing of epidermal growth factor receptor (EGFR) and Kristen-Rous sarcoma virus (KRAS) mutations in lung cancer by using cytology specimens. A recent article by the American Society of Clinical Oncology (ASCO) provided a provisional clinical opinion from expert consensus based on clinical evidence and literature available at the moment on the need for EGFR mutations testing in non– small cell lung cancer (NSCLC) patients. That consensus document addresses the clinical utility of EGFR mutation testing for patients with advanced NSCLC to predict response to first-line therapy with EGFR tyrosine kinase inhibitors (TKIs; erlotinib or gefitinib). Regarding the tissue to be used for molecular testing, the expert panel states that ‘‘[i]n order to obtain tissue for more accurate histologic classification or for investigational purposes, the Update Committee supports reasonable efforts to obtain more tissue than what is contained in a routine cytology specimen [emphasis added].’’ They also add that properly fixed material from cytology cell block preparations is generally required for analysis, as opposed to cytology smear preparations, and they believe that the development of more sensitive mutation analysis techniques will help in case of limited tumor material availability. Previous articles based on European expert panel meetings have come to similar recommendations. In our institution, most of the molecular testing performed on NSCLC samples corresponds to cytology specimens, mainly Papanicolaou smears and cell blocks. In accordance with Billah et al, in our experience, the use of endobronchial ultrasound (EBUS) for fine-needle aspiration results is extremely efficient for obtaining sufficient material for diagnosis and molecular testing. Thus, more aggressive and costly procedures for tissue sampling would in most cases be unnecessary. In addition, the availability of rapid on-site evaluation by our cytopathologists notably increases the efficiency of the procedure and the performance of the molecular analysis at our center. However, in the short term, the increasing tissue requirements for molecular testing (EGFR primary and secondary mutations, KRAS mutations, EML4-ALK fusion gene assessment, different genes’ copy number, and so forth) needed for clinical targeted-therapy decisions will ultimately define whether the tissue is still the issue. Finally, as the ASCO expert panel suggested, more sensitive mutation analysis techniques will be required to counterbalance limited tumor material availability.

Safety and Efficacy of Maintenance Therapy With a Nonspecific Cytochrome P17 Inhibitor (CYP17i) After Response/Stabilization to Docetaxel in Metastatic Castration-Resistant Prostate Cancer

BACKGROUND Frontline treatment of metastatic castration-resistant prostate cancer (mCRPC) consists of docetaxel-based chemotherapy. The median time to progression (TTP) from chemotherapy initiation is 6 to 8 months. Ketoconazole, a nonspecific cytochrome P17 inhibitor (CYP17i), blocks adrenal androgen synthesis. Low-dose ketoconazole (LDK), (200 mg three times daily [t.d.s]) has shown activity in mCRPC after progression to androgen deprivation. The role of a CYP17i after docetaxel treatment in the maintenance setting has been unexplored. METHODS We identified 38 patients with mCRPC who showed progression to luteinizing hormone releasing-hormone agonists (LHRHa) and who were treated with a median of 7 cycles of frontline three-weekly docetaxel (75 mg/m(2)) plus prednisone (10 mg/d) and LHRHa. Medical charts of 20 patients who showed no progression to docetaxel were reviewed. After the last docetaxel cycle, 10 patients received LDK maintenance treatment plus prednisone (10 mg/d) and LHRHa, whereas 10 patients received LHRHa alone. TTP was the primary endpoint. RESULTS After a follow-up of 27 months, disease in all patients receiving LHRHa alone progressed, whereas 8/10 patients progressed to maintenance therapy. Median TTP from docetaxel initiation was 11.5 months (95% confidence interval [CI], 6.3-16.6) for maintenance therapy and 9.2 months (95% CI, 8.5-9.9) for LHRHa alone (P = .047). The maintenance treatment was well tolerated. Only 1 patient experienced a grade 4 adverse event due to a nonsymptomatic pulmonary embolism. CONCLUSION This is the first study evaluating a CYP17i for maintenance therapy after docetaxel therapy. We showed a 2-month significant benefit in TTP for patients with mCRPC treated with LDK maintenance therapy after docetaxel, with a favorable toxicity profile. A large prospective randomized study using a CYP17i is warranted.

Genomic characterization of individuals presenting extreme phenotypes of high and low risk to develop tobacco‐induced lung cancer

Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome‐wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco‐induced non‐small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n = 3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (pcombined = 5.66 × 10−5; ORcombined = 2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (pcombined = 1.02 × 10−4; ORcombined = 2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early‐stage NSCLC. PDE10A and ATP10DmRNA expressions correlated with survival in 821 stage I–II NSCLC patients (p = 0.01 and p < 0.0001). PDE10A protein expression correlated with survival in 149 patients with stage I–II NSCLC (p = 0.002). In conclusion, we validated two variants associated with extreme phenotypes of high and low risk of developing tobacco‐induced NSCLC. Our findings may allow to identify individuals presenting high and low risk to develop tobacco‐induced NSCLC and to characterize molecular mechanisms of carcinogenesis and resistance to develop NSCLC.

Liquid Biopsies in Malignant Melanoma: From Bench to Bedside

Malignant melanoma is a malignant tumour originated from melanocytic cells and primarily involves the skin. However, it can also arise from the extracutaneous melanocytes located in the eye and mucosal surfaces. An increasing incidence of cutaneous melanoma in white population has been observed during the last decades. In Europe, the incidence is 10–15 new cases per 100,000 subjects annually and in the USA rises up to 18 cases per 100,000 inhabitants. However, the highest incidence has been reported in Australian and New Zealand population, with 40–60 cases per 100,000 inhabitants annually.

1078TiPPHASE II STUDY WITH IMMUNOTHERAPY WITH DENDRITIC CELLS (DC) COMBINED WITH INTRATUMORAL HILTONOL IN PATIENTS WITH ADVANCED CANCER

ABSTRACT Background: DC vaccines are active treatments for cancer and combination strategies are expected to increase anti-tumor activity. We explored the efficacy of intratumoral Hiltonol, a potent TLR3 agonist, combined with an autologous vaccine of DC loaded with self-tumor lysates that we developed in a previous study (Alfaro C, J Immunology 2011). Hiltonol is an stabilized form of polyI:C, a nucleic acid that mimics viral RNA. It induces local release of cytokines which promote inflammation, increase type I interferon secretion and stimulate leukocyte migration to infiltrate tissues. Preclinical data indicates that intratumoral Hiltonol activates pro-inflammatory changes that increase the efficacy of DC vaccination. Trial design: In this ongoing phase II study, 25 patients with advanced solid tumors non-amenable for conventional treatment are being treated with Hiltonol and DC vaccinations. Our vaccination protocol includes the following strategies: a) pretreatment with cyclophosphamide to decrease regulatory T cells; b) maturation and activation of DC with TNF-alpha, interferon-alpha and poly I:C, to induce type I interferon; c) use of autologous tumor as antigenic source to expose DC to antigens that are exclusive of tumor cells; and d) daily intradermal doses during four consecutive days in 2 cycles every 4 weeks. Two intratumoral ultrasound-guided injections of Hiltonol 0.25 mg are being administered on alternate days one week following each DC cycle. Sample size has been calculated using a two-stage Simons Minimax design, with alpha error a= 0.05 and beta-error = 0.10 for P0 = 0.05 and P1 = 0.25. The main objective is response rate. Secondary objectives include assessment of toxicity, overall survival and immunologic response (in vitro lymphocyte responses against tumor antigens; delayed hypersensitivity reactions; and assessment of DC maturation by expression of pro-inflammatory cytokines) (ClinicalTrials.gov Identifier: NCT01734564). This abstract was accepted and previously presented at the 2014 ASCO Annual Meeting Chicago, June 2014 (TPS3113). Disclosure: All authors have declared no conflicts of interest.

1076TiPRANDOMIZED PHASE II TRIAL WITH DENDRITIC CELL (DC) IMMUNOTHERAPY IN PATIENTS WITH COLORECTAL CARCINOMA AND LIVER METASTASIS FOLLOWING COMPLETE RESECTION AND ADJUVANT CHEMOTHERAPY

ABSTRACT Background: Cellular immunotherapy with DC has well-established anti-tumor activity, as confirmed by Sipuleucel-T, the first approved treatment based in this strategy. Preclinical and clinical data indicate that efficacy of DC immunotherapy is maximized when used in the setting of minimal residual disease. Our study explores the efficacy of an autologous DC vaccine loaded with self-tumor antigens that we developed in a previous study (Alfaro, J Immunology 2011) in patients with colorectal cancer that have undergone complete resection of hepatic metastasis and standard adjuvant therapy. Trial design: In this randomized phase II study, patients with colorectal carcinoma with hepatic metastasis that have undergone standard treatment, including complete surgical resection and standard adjuvant chemotherapy, are randomized to receive DC vaccine or to observation. The two-cycle vaccination protocol includes the following strategies: a) pretreatment with cyclophosphamide to decrease regulatory T cells; b) maturation and activation of DC with TNF-alpha, interferon-alpha and poly I:C, a potent inducer of type I interferon; c) use of autologous tumor from resected liver metastasis as antigenic source, to include antigens that are exclusive of tumor cells; and d) administration of daily intradermal vaccines during four consecutive days in 2 cycles every 4 weeks. Our aim is to replicate the immune response observed during an acute viral infection in terms of activation signals and persistence of antigens in lymph nodes. The main objective is progression-free survival. Thirty-six patients will be included, allowing to detect a HR = 1.75 (alpha error = 0.2, beta error = 0.35). Secondary objectives are: safety, overall survival and immunologic response (in vitro lymphocyte responses against tumor antigens; delayed hypersensitivity reactions; induction of tumor antibody responses; DC activation parameters including IL-12 and IL-6 production and expression of CD80, CD83, CD86, B7-H1, B7-H4 and B7-DC; assessment of DC maturation by expression of pro-inflammatory cytokines; and DC migration) (ClinicalTrials.gov Identifier: NCT01348256). This abstract was accepted and previously presented at the 2014 ASCO Annual Meeting Chicago, June 2014 (TPS3129). Disclosure: All authors have declared no conflicts of interest.