The role of CEA, CYFRA21-1 and NSE in monitoring tumor response to Nivolumab in advanced non-small cell lung cancer (NSCLC) patients.

BACKGROUND: CEA, CYFRA21-1 and NSE are tumor markers used for monitoring the response to chemotherapy in advanced adenocarcinoma, squamous cell carcinoma and small-cell lung cancer, respectively. Their role in cancer immunotherapy needs to be elucidated. METHODS: Patients with advanced non-small cell lung cancer (NSCLC) were treated with nivolumab 3 mg/kg every 2 weeks within the Italian Nivolumab Expanded Access Program. Blood samples were collected at baseline, at each cycle up to cycle 5 and then every two cycles until patient's withdrawn from the study. All patients underwent a CT-scan after every 4 cycles of treatment and responses were classified according to RECIST 1.1. The biomarkers serum levels were measured with a chemiluminescent microparticle immunoassay for CEA and with an immuno radiometric assay for CYFRA21-1 and NSE. The markers values at baseline and after 4 cycles were used to analyze the relationship between their variation over baseline and the tumor response, evaluated as disease control rate (DCR: CR + PR + SD), and survival (PFS and OS). RESULTS: A total of 70 patients were evaluable for the analysis. Overall, a disease control was obtained in 24 patients (35.8%, 4 PR + 20 SD). After 4 cycles of nivolumab a CEA or CYFRA21-1 reduction ≥ 20% over the baseline was significantly associated with DCR (CEA, p = 0.021; CYFRA21-1, p < 0.001), PFS (CEA, p = 0.028; CYFRA21-1, p < 0.001) and OS (CEA, p = 0.026; CYFRA21-1, p = 0.019). Multivariate analysis confirmed the ability of CYFRA21-1 reduction ≥ 20% to predict DCR (p = 0.002) and PFS (p < 0.001). CONCLUSION: The reduction in serum level of CYFRA21-1 or CEA might be a reliable biomarker to predict immunotherapy efficacy in NSCLC patients. NSE was not significant for monitoring the efficacy of nivolumab.

Sequential use of vinorelbine followed by gefitinib enhances the antitumor effect in NSCLC cell lines poorly responsive to reversible EGFR tyrosine kinase inhibitors.

Preclinical studies have suggested that combining cytotoxic agents with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) to treat EGFR-mutated tumors may increase their inhibitory effect depending on the order of drug administration. The antitumor efficacy of different treatment sequences using vinorelbine (VNB) and gefitinib (GEF) was investigated both in vitro and in vivo in non-small cell lung cancer (NSCLC) cell lines with the rationale of potentially translating these findings into the clinical setting. The EGFR-wild-type A549 and the EGFR-mutated (exon 21 L858R/exon 20 T790M) H1975 cell lines were treated as follows: GEF followed by VNB, VNB followed by GEF and the two drugs applied individually or concurrently. Results in vitro demonstrated that the sequence of VNB followed by GEF was significantly more active than single-agent treatments. The expression of activated EGFR and its downstream pathway genes indicated that the increased cytotoxic effect of the VNB and GEF treatment sequence was accompanied by inhibition of EGFR, AKT and ERK1/2. Moreover, the increased inhibition of tumor growth after treatment with VNB followed by GEF was also confirmed in CD1-nude mice that were xenotransplanted with H1975 cells (p < 0.0001). This effect was paralleled by a corresponding decrease in cancer glucose consumption, as assessed by micro-positron emission tomography scans (p < 0.05). These preclinical findings in NSCLC cell lines, which are poorly responsive to EGFR-TKIs, demonstrated that the sequential treatment of VNB followed by GEF induced a significant antitumor effect, which supports the translation of this treatment schedule into a clinical setting.

Role of microRNAs in malignant mesothelioma.

Malignant mesothelioma (MM) is an aggressive tumor, mainly derived from the pleura, which is predominantly associated with exposure to asbestos fibers. The prognosis of MM patients is particularly severe, with a median survival of approximately 9-12 months and latency between exposure and diagnosis ranging from 20-50 years (median 30 years). Emerging evidence has demonstrated that tumor aggressiveness is associated with genome and gene expression abnormalities; therefore, several studies have recently focused on the role of microRNAs (miRNAs) in MM tumorigenesis. miRNAs are small non-protein coding single-stranded RNAs (17-22 nucleotides) involved in numerous cellular processes that negatively regulate gene expression by modulating the expression of downstream target genes. miRNAs are often deregulated in cancer; in particular, the differential miRNA expression profiles of MM cells compared to unaffected mesothelial cells have suggested potential roles of miRNAs as either oncogenes or tumor suppressor genes in MM oncogenesis. In this review, the mechanism of MM carcinogenesis was evaluated through the analysis of the published miRNA expression data. The roles of miRNAs as diagnostic biomarkers and prognostic factors for potential therapeutic strategies will be presented and discussed.

Resistin is associated with overall survival in non-small cell lung cancer patients during nivolumab treatment.

PURPOSE: Since the role of resistin was evaluated only in patients with non-small cell lung cancer (NSCLC) not treated with immunotherapy, we aimed to evaluate levels of resistin during immunotherapy (nivolumab) and its prognostic role with regard to OS. METHODS/PATIENTS: From a cohort of 78 patients with advanced NSCLC enrolled in a prospective study at Ospedale Policlinico San Martino in Genoa (Italy), 43 patients have been considered for this sub-analysis because of the availability of samples. Before and during nivolumab administration, clinical information and blood samples were collected and resistin, matrix metalloproteinase (MMP)-8, MMP-9, and myeloperoxidase were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Median age was 71 with a prevalence of males and former smokers. Median resistin levels presented a peak at cycle 2 and then dropped down until the last cycle. Resistin correlated with all neutrophil degranulation products at cycle 1 (except for MMP-9) and at cycle 2 as well as with white blood cells and neutrophils. By a ROC curve analysis, a resistin value at cycle 2 of 19 ng/mL was tested as the best cut-off point for OS. Kaplan-Meier analysis demonstrated that patients above the resistin cut-off experienced a reduced OS (median OS 242.5 vs. 470 days, p = 0.0073), as confirmed by Cox proportional hazards regression analysis. CONCLUSIONS: Resistin levels > 19 ng/mL at the time of the second cycle of nivolumab treatment independently predict a reduced OS in patients with advanced NSCLC.

Evaluation of growth fractions with monoclonal antibodies to human alpha-DNA polymerase.

We have established and partially characterized a panel of monoclonal antibodies against alpha-DNA polymerase. One of the hybridomas, clone 5F, has been exploited for cell kinetic studies on three colon cancer cell lines, LOVO, SW 620, and SW 403, which are endowed with different growth patterns and differentiation status. By an immunoperoxidase method, we could demonstrate the specific intranuclear localization of alpha-DNA polymerase during the exponential phase of in vitro growth and contrast it with the diffuse distribution of the enzyme throughout the cytoplasm during the resting state. The percentage of intranuclear staining positive cells, evaluated at successive time points of in vitro growth, changed from 75 to 95% (assayed on Days 3 and 7) to 15 to 25% in confluent and resting populations assayed on Days 12 to 14. In agreement with the assumption that the enzyme moves from nucleus to cytoplasm after entering quiescence, alpha-DNA polymerase was still present in the cytoplasm or in the cytoplasmic perinuclear area of cells in resting phase cultures. Comparisons between traditional kinetic parameters (thymidine labeling index and primer-dependent alpha-DNA polymerase) and proliferative state determined by the monoclonal antibody supported the feasibility of this approach to define the proportion of actively proliferating elements in a tumor cell population. Moreover, parallel flow cytometric analysis performed on Days 5 and 14 of continuous culture showed fluctuations of alpha-DNA polymerase content in relation to exponential and steady-state phases, with a significant increase in the amount of alpha-DNA polymerase in actively proliferating populations and a progressive reduction of the enzyme as the cultures entered the resting stage.

Evidence for autocrine mitogenic stimulation by somatomedin-C/insulin-like growth factor I on an established human lung cancer cell line.

The production of immunoreactive somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) by the established cell line derived from a human lung carcinoma CALU-6 has been evidenced in the serum-free medium in increasing concentrations as a function of the incubation time. Gel filtration in acid conditions of cell-conditioned medium collected after 72 h showed peaks of immunoreactive Sm-C/IGF-I in the elution volume corresponding to the molecular weight of the synthetic Sm-C/IGF-I, and in the high molecular weight region, where specific binding sites for Sm-C/IGF-I could be also demonstrated. These results indicate that this established cell line produces high amounts of immunoreactive Sm-C/IGF-I and of Sm-C/IGF-I carrier protein. The pooled fractions corresponding to the molecular weight of synthetic Sm-C/IGF-I showed a competitive binding curve parallel to the standard in the Sm-C/IGF-I RIA system, and a mitogenic activity on cells from the same line similar to the one observed using two different pure Sm-C/IGF-I preparations, obtained by chemical synthesis or by DNA recombinant technology. When a monoclonal antibody (sm-1.2) raised against Sm-C/IGF-I was added into the medium, the mitogenic effect observed by both synthetic and cell-derived Sm-C/IGF-I peptide was completely abolished; the monoclonal antibody also partially inhibited the effect of 10% fetal calf serum and the thymidine incorporation observed in serum-free medium without growth factors. In serum-free medium the monoclonal antibody produced a 45% reduction of cells in S phase by thymidine labeling index without modification of the growth fraction as determined by primer-dependent alpha-DNA polymerase labeling index. In conclusion it seems that Sm-C/IGF-I has a critical role in the autocrine stimulation of the replication of this cell line.

Chemotherapy following estrogen-induced expansion of the growth fraction of human breast cancer.

We have evaluated the feasibility of a cytokinetically oriented regimen based on the induction of cell recruitment by diethylstilbestrol (DES) in locally advanced human breast cancer. Tumor proliferative activity was evaluated by the thymidine labeling index and the primer-dependent alpha-DNA polymerase labeling index, which gives an in vitro estimation of the growth fraction. Sixteen previously untreated patients received DES (1 mg daily for 3 days) followed by FAC [5-fluorouracil (600 mg/m2): Adriamycin (50 mg/m2): Cytoxan (600 mg/m2)] i.v. on day 4 every 21 days. Radical surgery was delayed to allow for three DES-FAC regimens in responsive patients. Proliferative activity on tumor biopsies was evaluated immediately before and after treatment with DES, 24 h after chemotherapy and, in nine patients, at the time of radical surgery. DES was able to induce a significant increase in thymidine labeling index in 8 of 16 patients, while the primer-dependent alpha-DNA polymerase labeling index was significantly increased in 13 of 16 tumors, independently of their estrogen receptor content. Subsequently administered chemotherapy induced an early decrease in tumor proliferation. In the nine patients submitted to surgery after three DES plus FAC courses, the average thymidine labeling index and primer-dependent alpha-DNA polymerase labeling index were 27.8 and 73% of the pretreatment values. Our preliminary results provide the rationale for the design of new therapeutic schemes in which antitumor drugs are given at the time of estrogen-induced tumor cell recruitment. Further extended studies are required to establish whether induction of tumor cell recruitment will actually translate into appreciable improvement of the clinical response to chemotherapy.

Vinflunine for the treatment of non-small cell lung cancer.

INTRODUCTION: Vinflunine belongs to the class of vinca alkaloids and acts by disrupting the microtubule dynamics during cell cycle; this agent is currently available for previously treated advanced transitional cell carcinoma in Europe. The aim of this invited review is to evaluate the potential role of vinflunine for the treatment of non-small cell lung cancer (NSCLC). Areas covered: The potential role of vinflunine in NSCLC is discussed on the basis of the available data, including full papers and meeting abstracts. Relevant preclinical studies describing the pharmacological properties of vinflunine are also included. The review also summarizes clinical studies, including phase I trials involving NSCLC among other tumors as well as phase II/III trials specifically addressing this malignancy. Additionally, the safety profile and the current regulatory status of vinflunine is discussed. Expert opinion: Vinflunine is active as single agent and as part of platinum-based combinations in NSCLC. It results non-inferior to docetaxel in a randomized phase III trial including previously treated NSCLC patients; additionally, its safety profile is generally considered manageable. Ultimately, further studies are needed to confirm the role of vinflunine in NSCLC, in consideration of the evolving evidence regarding targeted therapies and immune check-point inhibitors.

Conventional versus cytokinetic polychemotherapy with estrogenic recruitment in metastatic breast cancer: results of a randomized cooperative trial.

Diethylstilbestrol (DES) can induce a recruitment into the proliferative pool of previously resting breast cancer cells in vivo. In order to verify if estrogenic recruitment could result in a larger tumor cell killing by chemotherapy, 117 patients with metastatic breast cancer were randomized to receive CEF (cyclophosphamide, 600 mg/m2; epidoxorubicin, 60 mg/m2; and 5-fluorouracil, 600 mg/m2 on day 1); DES-CEF (cyclophosphamide, 600 mg/m2 on day 1; DES, 1 mg orally on days 5, 6, and 7; and epidoxorubicin, 60 mg/m2, and 5-fluorouracil, 600 mg/m2, on day 8) every 21 days. No significant difference in objective response rates, survival, or progression-free survival was seen between the two regimens. Patients in the DES-CEF arm experienced a higher complete response (CR) rate (24.1% v 16.1%), which reached statistical significance in the case of soft-tissue metastasis (48% v 27.3%; P less than .05) and estrogen receptor-negative tumors (35.7% v 11.1%; P less than .025). Survival and progression-free survival of patients refractory to treatment were not worsened by estrogenic recruitment. In the subset of patients failing after adjuvant polychemotherapy, DES-CEF unexpectedly induced a significantly longer survival (greater than 802 days v 375 days; P = .029) and progression-free survival (239 days v 192 days; P = .041) than CEF. The DES-CEF regimen was more myelotoxic, and 43.3% of the DES-CEF cycles had to be delayed because of leukopenia in comparison with 11.8% of the CEF cycles (P less than .0001). In conclusion, chemotherapy with estrogenic recruitment was able to induce more CRs in certain subsets of patients and a significant prolongation in survival and progression-free survival of patients failing after adjuvant polychemotherapy. These results have been achieved despite a significantly lower dose intensity of chemotherapy.

Increased cyclin D1 expression is associated with features of malignancy and disease recurrence in ovarian tumors.

Alterations in the expression of cyclin D1 have been reported frequently in several human cancers, but their significance in the multistep model of carcinogenesis has been scantly described. To define the pattern of cyclin D1 expression in the development of ovarian cancer and clinical outcome, 55 cases of benign ovarian tumors, 12 borderline cases, and 37 ovarian carcinomas (32 primary and 5 recurrent carcinomas) were studied. Analyses were carried out on fresh tumor specimens by Western blotting and reverse transcription-PCR and provided significant superimposable results (P = 0.00001). Cyclin D1 abundance was classed according to the densitometric values as undetectable, detectable, well detectable, and highly detectable. A significant increase (P < 0.000001) in median cyclin D1 values was observed from benign (0.038; range, 0.001-0.705) to borderline (0.226; range, 0.001-0.623) to malignant (0.347; range, 0.027-2.330) to recurrent (0.887; range, 0.309-2.2260) tumors. In addition, higher median cyclin D1 values were reported in serous carcinomas (P = 0.058) and advanced-stage diseases (P = 0.003). Survival analyses carried out in the 32 primary carcinomas showed no significant difference in overall survival between detectable versus well/highly detectable cyclin D1 neoplasms. Conversely, a significant relationship between cyclin D1 expression and progression-free survival was found (P = 0.031). These results may elucidate the function of altered cyclin D1 expression in ovarian tumorigenesis and provide a basis for additional studies on its prognostic role.

Tumour kinetics, response to chemotherapy and survival in primary ovarian cancer.

The analysis of thymidine labelling index (TLI) in relation to clinico-pathological variables and survival was carried out in 111 ovarian cancer patients. The significance of TLI in predicting response to aggressive first line chemotherapy regimens was examined. The overall median TLI value of 1.8% was used as a cut-off to discriminate slowly from highly proliferating cancers. 94 patients entered into two consecutive randomised trials, and were treated with six courses of cisplatin-based chemotherapy with or without doxorubicin. A significantly higher objective response of 60% was reported in the subset of patients with TLI > 1.8% as compared to 35% in patients with TLI < or = 1.8% (P = 0.03). In addition, patients achieving complete response had tumours with median TLI of 3.8% as compared to 2.4% for partial responders, 1.5% for patients with stable disease and 1.7% for those with progressive disease. A significant increase in tumour kinetics was observed in advanced cancers (P = 0.001), more undifferentiated tumours (P = 0.02) and postsurgical residual disease greater than 2 cm (P = 0.04). In univariate analysis, TLI failed to influence significantly clinical outcome: 26 versus 32 months median survival time for patients with high and low tumour TLI, respectively. In the Cox's regression model, the only independent prognostic variables were performance status and amount of residual disease after primary surgery (P = 0.000).

Prediction of survival by thymidine labelling index in patients with resistant ovarian carcinoma.

The relationship between tumour proliferative activity, evaluated by thymidine labelling index (TLI), clinicopathological variables and clinical outcome, was analysed in a series of 64 chemotherapy-resistant, ovarian cancer patients. The median TLI of 4.6% (range 0.01-45.7) was used as the cut-off to discriminate rapidly from slowly proliferating tumours. Univariate analyses showed a significant advantage in survival for patients with TLI less than or equal to 4.6 (P = 0.0004), ECOG performance status less than or equal to 1 (P = 0.0001) and residual disease after primary surgery less than or equal to 2 cm (P = 0.019). Multivariate analysis demonstrated that performance status was the only independent prognostic variable, although TLI was the last covariate removed from the Cox's regression model.

Thymidine labelling index as prognostic factor in resected non-small cell lung cancer.

To assess the prognostic value of tumor proliferative activity, 89 patients with operable non-small cell lung cancer were studied. Tumor samples were obtained during surgery and cell kinetics were analyzed by the in vitro thymidine labelling index (TLI). The overall median TLI (2.9) was used to identify two subsets of patients with high and low proliferating tumors. In univariate analysis survival was significantly longer in patients with lower TLI (P = 0.047) and with stage I-II (P = 0.003) and T1-T2 tumors (P = 0.043). In multivariate analysis, stage was the most important prognostic parameter (P = 0.004). The risk of death for patients with TLI higher than 2.9 was increased (hazard ratio = 2.01, CI = 0.96-4.27).

Timed sequential chemotherapy following ifosfamide-induced kinetic recruitment in refractory ovarian cancer.

A total of 22 relapsed or refractory ovarian cancer patients were treated with ifosfamide-containing polychemotherapy. Kinetic analyses were done to evaluate the tumor-cell-recruiting potential of the alkylating agent. Our study shows that ifosfamide can enhance tumor proliferative activity in pretreated ovarian cancers.

Feasibility of thymidine labelling index on fine needle aspirates from breast cancer: comparison with surgical samples.

Fine-needle aspiration (FNA) provides a suitable diagnostic tool in the management of patients with breast cancer lesions. The current study reports on tumor proliferative activity, by 3H-Thymidine Labelling Index (TLI), assessed on 59 FNA (TLI1) and 28 surgical specimens (TLI2) from the same breast cancer patients. Median TLI values from FNA and surgical material were 1.0% and 0.7%, respectively. In the 28 patients, evaluable for the comparison between TLI1 and TLI2, the association was found to be highly significant (p = 0.000). Moreover, no change in tumor proliferative activity was observed in the majority (79%) of cases when evaluated preoperatively and at surgery. This study confirms the feasibility of TLI analysis on FNA from breast cancer and provides results superimposable on those obtained in a tissue sample from the same patient.

Cytotoxicity in vitro and preliminary antitumor activity in vivo of a novel organotin compound.

The cytotoxic effect and antitumor activity induced by the novel organotin compound triethyltin(IV)lupinyisulfide hydrochloride, have been investigated. Different patterns of antiproliferative effects have been observed in a panel of human tumor cell lines in vitro. Toxicity studies in mice reported acute toxicity at the doses of 21 and 17.5 mg/kg which progressively disappeared at lower concentrations. On this basis, the doses of 3.5, 7 and 14 mg/kg were selected to assess the antitumor activity in vivo against the P388 leukemic cells xenografted in mice. This compound was able to induce a dose-dependent significant reduction of tumor volume, up to 46%, at the highest concentration (p = 0.0062) without important toxicity, as also confirmed by histological analysis of the main organ tissues. This preliminary study seems to hold interest for further investigations in different tumor models as well as for the evaluation of optimal drug route and schedule.

Manipulation of the growth rate of human breast cancer cells by antiandrogen followed by chemotherapy.

The effects induced by the antiandrogen Cyproterone Acetate (CPA) on the proliferation of EVSA-T human breast cancer cells endowed with androgen receptors were studied. Kinetic analyses were carried out by two autoradiographic techniques measuring the percentage of cells in S-phase and the growth fraction (GF). The exposure of the cultures to CPA for 24 h caused a marked inhibition on S-phase cells without significantly affecting the CF. The accumulation of cells in G1-phase, confirmed by cytometric analysis, was rescued to the S compartment by replacing the culture medium 24 h after CPA administration. Exposure of EVSA-T to Doxorubicin or Methotrexate after CPA and medium change at the time of maximal proliferative recruitment, led to an enhancement of cytotoxicity as demonstrated by colony survival assay.

Monoclonal antibodies to alpha DNA polymerase as a marker of cell proliferative activity.

A hybridoma cell line (5F) secreting monoclonal antibodies directed to alpha DNA polymerase has been developed. Kinetic studies on peripheral blood lymphocytes stimulated with mitogen and human colon cancer cell lines established in vitro were made by the two autoradiographic techniques of Thymidine Labelling Index and Primer-dependent alpha DNA polymerase Labelling Index and the immunoperoxidase assay (PAP) with monoclonal antibody to alpha DNA polymerase. We demonstrated the exclusively intranuclear presence of alpha DNA polymerase in lymphocytes induced to proliferate and actively growing colon cancer cells in contrast with the cytoplasmic distribution of the enzyme in resting stage populations. The feasibility of using monoclonal antibodies to alpha DNA polymerase to determine cell growth fraction was evaluated.

Antiproliferative activity and interactions with cell-cycle related proteins of the organotin compound triethyltin(IV)lupinylsulfide hydrochloride.

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.

In vivo cytokinetic effects of recombinant human growth hormone (rhGH) in patients with advanced breast carcinoma.

We have investigated the possibility of inducing a kinetic recruitment of breast cancer cells by the in vivo administration of recombinant human Growth Hormone (rhGH). Twelve patients with advanced breast cancer received rhGH i.m. for 2 days immediately before the first course of chemotherapy. The following biological parameters have been evaluated before and 24 hours after rhGH administration: tumor TLI, tumor IGF-I content, serum IGF-I concentration. The mean tumor TLI values before and after rhGH were 1.3% and 2.6% respectively; median tumor and serum IGF-I levels before rhGH were 4.64 ng/g and 63.5 ng/ml respectively; after the administration of rhGH median tumor IGF-I content was 1.8 and median serum IGF-I level was 112 ng/ml. These data suggest that, in vivo, rhGH stimulates breast cancer cell proliferation; the mitogenic stimulus is likely due to the local production of IGF-I induced by rhGH.