Efficacy Of Targeted Therapies Research Articles

Retrospective Analysis of Fifth-Line Targeted Therapy Efficacy in Patients with Metastatic Renal Cell Carcinoma

Objectives: Current evidence of sequence-targeted therapy (TT) for patients with metastatic renal cell carcinoma (mRCC) beyond fourth-line is sparse. The aim of this study was to describe the efficacy and toxicity of fifth-line TT in patients with mRCC. Methods: Out of 406 patients treated in first-line, 25 patients (6.16%) with more than 4 lines of TT were retrospectively reviewed at a German academic high-volume cancer center. Response was assessed by the use of standard Response Evaluation Criteria in Solid Tumors version 1.0, and toxicity was graded according to the Common Toxicity Criteria for Adverse Events version 3.0. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Cox proportional hazard models were applied to explore predictors of PFS and OS in univariable and multivariable analyses. Results: Disease control rate for fifth-line treatment was 20%. Median OS from the beginning of first-line therapy was 50.2 months (IQR (interquartile range) 38.9-76.7). Median OS from the time of initiation of fifth-line therapy was 6.2 months (IQR 3.1-23.8). Median PFS for fifth-line TT was 4.1 months (IQR 1.81-9.07) and did not correlate to treatment response in first-line TT. Conclusions: Highly selected patients might benefit from fifth-line treatment independently from treatment response in first-line TT.

Expression of Forkhead box M1 in soft tissue leiomyosarcoma: Clinicopathologic and invitro study using a newly established cell line.

Leiomyosarcoma (LMS) of soft tissue is a sarcoma with smooth-muscle differentiation, and conventional chemotherapy does not improve its outcome. The application of novel antitumor agents and precise prognostication has been demanded. The expression of the protein Forkhead box M1 (FOXM1), a member of the FOX family, is considered an independent predictor of poor survival in many cancers and sarcomas. However, the expression status of FOXM1 in LMS is poorly understood. The purposes of this study were to examine the correlation between the expression of FOXM1 and clinicopathologic or prognostic factors and to clarify the efficacy of FOXM1 target therapy in LMS. We evaluated the immunohistochemical expressions of FOXM1 using 123 LMS tumor specimens. Univariate and multivariate survival analyses revealed that FOXM1 expression was associated with poor prognosis in LMS. An invitro study was then carried out to examine the antitumor effect of a FOXM1 inhibitor (thiostrepton) and siRNA on a novel LMS cell line, TC616. We also assessed the efficacy of the combined use of doxorubicin and thiostrepton. Thiostrepton showed dose-dependent antitumor activity and TC616 cells treated with the combination of thiostrepton and doxorubicin showed lower proliferation compared to those treated with either drug individually. FOXM1 interruption by siRNA decreased cell proliferation and increased chemosensitivity. In conclusion, FOXM1 has potential to be a therapeutic target for LMS.

Third-line therapy in metastatic renal cell carcinoma (mRCC): Results from the International mRCC Database Consortium (IMDC).

e15578 Background: Third line targeted therapy efficacy in mRCC is not well characterized and is not reimbursed in many jurisdictions worldwide. Methods: The IMDC consists of consecutive patient se...

Clinical study of targeted therapy in the treatment of metastatic non–clear cell renal cell carcinoma

Objective To evaluate the efficacy and side effects of targeted therapy in the treatment of metastatic non–clear cell renal cell carcinoma (nccRCC). Methods Data of patients with metastatic renal cell carcinoma (RCC) who received sorafenib (400 mg, twice one day) or sunitinib (50 mg/d, 4 weeks on/2 weeks off) as the first–line targeted therapy from Dec. 2006 to May. 2014 were analyzed retrospectively. Pathological diagnosis indicated 317 clear cell RCC (ccRCC) and 56 nccRCC (29 papillary RCC and 27 other subtypes). Kaplan–Meier method was used to assess overall survival (OS), and log–rank test was performed to compare the difference of median follow–up between the two groups. Side effects were also documented and analyzed in all the patients. Results The follow–up period was 2.7 to 79.9 mon (median: 34.3 mon) in the whole group, among which the median follow–up was 36.6 mon in metastatic ccRCC patients and 18.5 mon in nccRCC patients, respectively. The one, two and three–year survival rate in metastatic ccRCC patients were 90.2%, 47.9% and 24.6%, respectively. The one–year survival rate in metastatic nccRCC patients was 60.7%, and only one patient survived over two years. Compared with ccRCC patients, those with nccRCC showed poorer prognosis (P 0.05). Conclusions Targeted therapy is efficient in the treatment of metastatic nccRCC patients and the side effects are reversible, although the prognosis is poorer compared with ccRCC. Key words: Metastatic renal cell carcinoma; Clear cell renal cell carcinoma; Non–clear cell renal cell carcinoma; Targeted therapy

Third-line therapy in metastatic renal cell carcinoma: Results from the International mRCC Database Consortium.

430 Background: Third-line targeted therapy efficacy in metastatic renal cell carcinoma (mRCC) is not well characterized and many funding bodies do not provide reimbursement for it. Methods: The International mRCC Database Consortium (IMDC) consists of consecutive patient series from 25 cancer centers. It was queried for specific sequences of targeted therapy and third-line therapy. Kaplan Meier estimates were used for survival. Cox proportional hazards models were used to adjust hazard ratios for confounders. Patients that stopped second-line therapy were divided into two groups: those that went onto third-line therapy and those did not. Results: 4,050 patients were treated with first-line targeted therapy, of which 2,011 (49.6%) had second-line therapy and 879 (21.7%) had third-line targeted therapy. The most common third-line therapies were everolimus 25%, sorafenib 14%, sunitinib 13%, temsirolimus 11%, pazopanib 10%, and axitinib 6%. IMDC prognostic groups at third-line therapy initiation were 6% favorable risk, 67% intermediate risk, and 27% poor risk. Overall response rate for third-line therapy was 10.5% and 50.9% had stable disease in those patients that were evaluable. Median PFS was 5.1 months (95% CI, 4.5-5.7) and median OS from third-line therapy initiation was 12.0 months (95% CI, 10.7-12.9). Patients stopping second-line therapy that move on to third-line therapy vs. those that do not receive third line therapy have a median OS from stopping second-line therapy of 13.1 vs. 2.3 mons (p<0.0001). When adjusted for second-line IMDC prognostic criteria and KPS at second-line treatment cessation, patients who do receive third-line therapy have a HR of death of 0.41 (95% CI, 0.32-0.52; p<0.0001) compared to those that do not receive third-line therapy. This may be in part due to patient selection. To further limit bias, when excluding patients that live less than 3 months after second-line therapy cessation, the adjusted HR was similar. Conclusions: Third-line targeted therapy has demonstrated activity and is prevalent in use. Further studies are required to determine appropriate sequencing.

Efficacy of targeted therapies after PD1/PD-L1 inhibitors in metastatic clear cell renal cell carcinoma (mRCC): A multi-institution retrospective cohort.

456 Background: Programmed death-1 (PD-1)/PD-1 ligand (PD-L1) inhibitors showed activity in mRCC and are currently in development in first-line and previously treated patients (pts). Whether these drugs will modify efficacy of subsequent targeted therapy (TT) including VEGFR tyrosine kinase inhibitors (TKI) or mTOR inhibitors is unknown. Methods: Medical records of RCC patients treated with investigational PD-1 or PD-L1 inhibitors who received subsequent treatment with TT were reviewed in 4 institutions. Baseline characteristics at the time of subsequent therapy and outcome data including time to treatment failure (TTF), best response, 1 and 2 year overall survival (OS) were retrospectively collected. Results: Of 89 pts who received PD-1/PD-L1 inhibitors, 56 patients (M/F 39/17, clear-cell 53, IMDC good 3/intermediate 16/poor 11/unknown 26) have received a subsequent therapy after PD-1/PD-L1 blockade, while 7 patients are still on therapy and 26 patients did not receive subsequent therapy. Among these 26 patients, 12 died from disease and 14 are still alive off-systemic therapies. 43 pts received VEGFR TKI and 13 received mTOR inhibitors as first subsequent TT. Median follow up from start of the subsequent TT is 16.1 months (range: 0.2, 30.6 months). TT post PD-1/PD-L1 was administered as second line in 9 patients (16%), third line in 24 patients (43%), > fourth line in 23 patients (41%). Median TTF was 6.9 months (range: 0.2+, 23.0), and was 6.9 and 5.7 months in patients who received VEGFR TKI and mTOR inhibitors respectively. One-year and 2-year OS from the initiation of subsequent TT was 58% (95% CI: 41-72%) and 36% (95% CI: 18%-54%), respectively. Investigator-assessed best response to subsequent TT was evaluated in 53 out of 56 patients: PR (n=7, 13%), SD (n=33, 62 %), and PD (n=13, 25%). Conclusions: This is the first report of TT efficacy after PD-1/PD-L1 inhibition. In this selected population, median TTF suggests a sustained benefit of both VEGFR TKI and mTOR inhibitors after PD-1/PDL1 inhibition.

Safety and efficacy analysis on modified FOLFOXIRI as first-line treatment for advanced colorectal cancer in China-single center experience

To evaluate the safety and preliminary efficacy of modified FOLFOXIRI (combination of reducing dosage irinotecan, oxaliplatin and fluorouracil) in first-line treatment for patients with metastatic colorectal cancer. A total of 53 patients with advanced colorectal cancer receiving modified FOLFOXIRI regimen were recruited continuously from January 2010 to January 2014. Safety profile was recorded based on NCI Common Terminology Criteria for Adverse Events version 3.0 (NCI-CTCAE v3.0). Objective response was evaluated by Response Evaluation Criteria in Solid Tumors version1.1 (RECIST 1.1) after administration of at least 4 cycles chemotherapy. Kras and Braf gene sequencing was tested by dideoxy chain-termination method. Relation between efficacy and two genes was examined. Among 53 patients, no treatment-related mortality was presented. The rate of grade 3 to 4 adverse event was 32.1% (17/53), including neutropenia 13.2%(7/53), anemia 11.3% (6/53) and fatigue 9.4% (5/53). Overall response rate (ORR) and disease control rate (DCR) were respectively 65.9% (29/44) and 90.0% (40/44). Radical resection rate (R0) was 29.5% (13/44). Efficacy of mFOLFOXIRI regimen plus targeting therapy was assessed in 44 patients. mFOLFOXIRI regimen plus targeting therapy achieved an ORR of 72.7% (8/11), which was higher than the ORR 65.9% (21/33) of triplet regimen alone, but the difference was not statistically significant (P=0.198). Paraffin specimens of 48 colorectal cancer cases were tested. Twenty-one cases were Kras mutant (43.75%), 3 cases were Braf mutant (6.25%). There were no significant differences between two groups (P>0.05). Reducing dosage mFOLFOXIRI can be safely used in advanced colorectal cancer and can achieve promising results in terms of short term efficacy.

Chemotherapy and molecular targeted therapy of advanced gastric cancer

Chemotherapy and molecular targeted therapy are the main treatments in unresectable gastric cancer. In recent years,due to the adjustment and optimization of chemotherapy regimens which are consist of fluorouracil,platinum and taxane,the survival time of gastric cancer patient has been prolonged and the quality of life has been improved. The molecular targeted therapy in gastric cancer arouses more and more concern,and there are many phrase Ⅲ clinical trials which are used to assess the efficacy of molecular targeted therapy. The advances of chemotherapy and molecular targeted therapy bring new hope to advanced gastric cancer patients. Key words: Stomach neoplasms; Drug Therapy; Molecular targeted therapy

Relapsed small cell lung cancer: treatment options and latest developments

According to recent analyses, there was a modest yet significant improvement in median survival time and 5-year survival rate of limited stage small cell lung cancer (SCLC) in North America, Europe, Japan and other countries over the last 30 years. The median survival time of limited stage SCLC is 15-20 months and 5-year survival rate is 15% or less. In terms of extensive stage SCLC, a median survival time of 9.4-12.8 months and 2-year survival of 5.2-19.5% are still disappointing. Despite being highly sensitive to first-line chemotherapy and radiotherapy treatments, most patients with SCLC experience relapse within 2 years and die from systemic metastasis. While several clinical trials of cytotoxic chemotherapies and molecular targeting agents have been investigated in the treatment of relapsed SCLC, none showed a significant clinical activity to be able to exceed topotecan as second-line chemotherapy. There are problematic issues to address for relapsed SCLC, such as standardizing the treatment for third-line chemotherapy. Topotecan alone was the first approved therapy for second-line treatment for relapsed SCLC. Amrubicin is a promising drug and a variety of trials evaluating its efficacy have been carried out. Amrubicin has shown superiority to topotecan in a Japanese population, but was not superior in a study of western patients. There are some controversial issues for relapsed SCLC, such as treatment for older patients, third-line chemotherapy and efficacy of molecular targeting therapy. This article reviews current standard treatment, recent clinical trials and other topics on relapsed SCLC.

Why anti-Bcl-2 clinical trials fail: a solution

The alteration in expression of B cell lymphoma-2 (Bcl-2) family of protein members in cancer is involved mainly in the regulation of apoptosis. Bcl-2 family proteins are currently used as major targets in the development of methods to improve treatment outcomes for cancer patients that underwent clinical trials. Although many agents have been developed for targeting Bcl-2 in the past decade, some previous attempts to target Bcl-2 have not resulted in beneficial clinical outcome for reasons unknown. Here, we propose that this was due in part for not considering the cellular level of a different antiapoptotic protein, i.e., galectin-3 (Gal-3). Gal-3 is a member of the β-galactoside binding protein family and a multifunctional oncogenic protein which regulates cell growth, cell adhesion, cell proliferation, angiogenesis, and apoptosis. Gal-3 is the sole protein that contains the NWGR anti-death motif of the Bcl-2 family and inhibits cell apoptosis induced by chemotherapeutic agents through phosphorylation, translocation and regulation of survival signaling pathways. It is now established that Gal-3 is a candidate target protein to suppress antiapoptotic activity and anticancer drug resistance. In this review, we describe the role and relevance of Gal-3 and Bcl-2 protein family in the regulation of apoptosis and propose a novel combination therapy modality. Combination therapy that targets Gal-3 could be essential for improvement of the efficacy of Bcl-2 targeting therapy in cancers and should be studied in future clinical trials. Otherwise, not considering Gal-3 cellular level could lead to trial failure.

Targeted therapies (TT) in metastatic renal cell carcinoma (mRCC): An Italian survey of 902 pts.

e15514 Background: TT have improved the outcome in mRCC; however, definite data regarding their efficacy when used in the community setting are still insufficient. This survey was aimed to evaluate survival in subsequent lines of treatment (tx) in mRCC pts treated outside clinical trials. Methods: Individual data from the clinical records of 902 pts treated with TT from mid 2007 to December 2012 were obtained from 28 Italian Institutions, through a questionnaire sent after approval by local ethical committees. Results: Median follow up was 26 mo (range 1-85), median age was 60 yrs (range 25-89), 75% were male. At onset metastatic sites were lung (62%), lymph nodes (38%), bone (31%); each of the other sites <10%. Median overall survival (mOS) was 24 mo (range 1-85) without statistical difference among the various centres. Sunitinib was the 1stline tx in 693 pts (76.6%), sorafenib in 124 (13.7%) , temsirolimus in 35 (4%), bevacizumab in 26 (3%), pazopanib in 19 (1.5%), others 5. Median PFS of 1stline (mPFS1) sunitinib was 11 mo and that of sorafenib 7 mo. Type of response: CR 32 (3.5%), PR 274 (30%), SD 275 (30%); PD 265 (29%), not evaluable 33 (3.5%), tx ongoing 23 (2.5%). Dose reduction of sunitinib and sorafenib was required in 49% and 39% of the cycles administered, respectively; 137 pts (15%) received only 1 cycle of sunitinib mostly for rapid progression and/or deterioration. 2ndline tx was performed in 46% of the pts: sorafenib 143 (16%), everolimus 121 (13%), sunitinib 100 (11%), temsirolimus 25 (3%), chemo-immunotherapy 26 (3%), others 1%. mPFS2 was 6 mo with sunitinib and about 4 mo with the other tx (p=0.001). 155 pts (17%) received 3 lines of tx: mOS of TKI-TKI-mTOR (A), TKI-mTOR-TKI (B) and other (C) triplets were 48, 37 and 33 mo, respectively; A vs B p= 0.06; A vs C p= 0.01. Overall PFS3 was 6 mo. Conclusions: i) This survey shows that data of clinical trials were reproduced in the real world setting; ii) a good proportion of pts achieved long OS after subsequent lines of tx; iii) on the other hand, a similar percentage of pts experienced rapid progression; iv) further studies are needed to better identify the different subsets of pts in order to improve the efficacy of TT.

Use of phosphoprotein profiling with high-density fluorescence protein microarray to reveal a response predictor to a multikinase inhibitor sorafenib.

e15080 Background: The efficacy of sorafenib in patients with unresectable hepatocellular carcinoma (HCC) has been revealed in the recent phase III SHARP (Sorafenib HCC Assessment Randomized Protocol) clinical trial, but only a small proportion of patients achieved the anticipated therapeutic outcomes. It is essential to identify biomarkers that will discriminate such patients. Methods: We newly developed high-density fluorescence reverse-phase protein arrays and used them to profile the phosphorylation status of 180 signaling molecules in the 120 pathways registered on the NCI-Nature curated database in 23 HCC cell lines with different sensitivities to sorafenib. Results: Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residues 235/236 (p-rpS6 S235/236) was the most significantly correlated with the sensitivity of HCC cells to sorafenib. The high expression of p-rpS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) and/or the mitogen-activated protein kinase (MAPK) pathways downstream of c-RAF. Conclusions: The present results indicate that p-rpS6 S235/236 is a potential predictor of unresponsiveness to sorafenib. Cross-talk between the mTOR and MAPK signaling pathways in sorafenib-resistant HCC cell lines was also revealed. A combination of mTOR and MAPK inhibitors may improve the efficacy of molecular targeting therapy against HCC.

Prognostic significance of Bcl-xL expression and efficacy of Bcl-xL targeting therapy in urothelial carcinoma

Background:Bcl-xL has an important role in the control of cell death through its inhibition of apoptosis. The aim of this study was to investigate the clinicopathological significance of Bcl-xL in upper urinary tract urothelial carcinoma (UTUC) and the therapeutic effect of targeting Bcl-xL protein in urothelial carcinoma (UC) cells.Methods:We evaluated the immunohistochemical expression of Bcl-xL in 175 UTUC patients to determine the clinical role of Bcl-xL expression in clinical outcome. We used bafilomycin A1 (BMA) as a specific inhibitor of Bcl-xL to examine the biological effects in UC cells in vitro and in vivo.Results:Immunohistochemical analysis of Bcl-xL expression revealed that patients with a high Bcl-xL score had a significantly lower 5-year cancer-specific survival (CSS) rate (53.2%) than those with a low Bcl-xL score (77.2%) (P=0.0011). Multivariate analysis indicated that a high Bcl-xL score was an independent prognostic factor of CSS (P=0.023). BMA inhibited UMUC-3 cell proliferation in vitro by induction of apoptosis. Treatment with BMA significantly inhibited tumour growth in UMUC-3 tumours in this mouse xenograft model accompanied by an elevated apoptosis induction.Conclusion:Bcl-xL appears to be a significant molecular marker for the prognosis of UTUCs. Targeting Bcl-xL may be a promising therapeutic strategy for patients with UC.

Abstract 4654: Genomic and proteomic profiling of signaling pathways targeted by a multi-kinase inhibitor, sorafenib.

Abstract Background: Deregulation of intracellular signaling by various genetic alterations is a hallmark of cancer. Therapeutics targeting such activated signaling pathways have achieved significant progress, but the precise pathway(s) targeted by sorafenib has remained unclear. Methods: Using high-density reverse-phase protein arrays, we profiled the phosphorylation status of 180 signaling molecules in the 120 pathways registered on the NCI-Nature curated database in 23 hepatocellular carcinoma (HCC) cell lines showing different sensitivities to sorafenib. We also sequenced the entire coding regions of 518 protein kinase genes in 20 overlapping HCC cell lines. Results: We identified 25 non-synomymous alterations in the 4 mTOR and MAPK pathway genes (RPS6KA1, RPS6KA6, RPS6KB2, and BRAF) in HCC cell lines 1 to 11. We also found that the levels of phosphorylated S6 ribosomal protein and c-Raf were associated with the sensitivity of the HCC cell lines to sorafenib. A mTOR inhibitor, AZD8055, exerted growth-inhibitory activity against sorafenib-resistant HCC cell lines. Discussion: Although a recent phase III clinical trial has demonstrated the effectiveness of sorafenib in patients with unresectable HCC, only a small proportion of the patients achieved the anticipated therapeutic benefits. The present results indicate cross-talk between the mTOR and MAPK signaling pathways in sorafenib-resistant HCC cell lines. Use of a combination of mTOR and MAPK inhibitors may improve the efficacy of molecular targeting therapy against HCC. Citation Format: Mari Masuda, Kazufumi Honda, Tesshi Yamada. Genomic and proteomic profiling of signaling pathways targeted by a multi-kinase inhibitor, sorafenib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4654. doi:10.1158/1538-7445.AM2013-4654

Neoadjuvant treatment in advanced renal cell carcinoma: current situation and future perspectives

Neoadjuvant approaches in renal cell carcinoma are currently under investigation, following the demonstration of targeted therapy efficacy in the metastatic setting. It raises the issues of downsizing locally advanced or nonresectable tumor and offering organ-sparing surgery, saety and its potential role in early micrometastatic disease. Relevant studies of the neoadjuvant setting in renal cell carcinoma with targeted therapies were identified from the literature, clinical trial databases and conference abstracts. To date, a neoadjuvant approach appears feasible in terms of saety. Currently available drugs do not achieve major tumor downsizing with primary tumor diameters response rate of 10%. Neoadjuvants should only be considered in clinical trials or as a litmus test in locally advanced patients.

JS1-1 - Biomarker-Based Cancer Therapy Personalization

ABSTRACT The efficacy of cancer treatments varies significant from case to case, even if their tumors are categorized into the same histological subtypes and stages. Thus it would be necessary to identify a biomarker that could provide evidence about the probability of benefit or toxicity from a specific therapeutic intervention. EGFR gene mutation, for example, is a predictive biomarker for epidermal growth factor receptor tyrosine kinase inhibitors, but its identification is a rare successful case and only a limited number of predictive biomarkers are currently available for cancer therapeutics. Technologies such as microarray, high-speed sequencing, and mass spectrometry have advanced rapidly in recent years, allowing comprehensive analyses of the cancer genome and proteome. It is anticipated that the application of these technologies will contribute to the discovery of new cancer biomarkers. Antibody-based proteomics is a concept that emerged in alliance with the progression of genome-wide antibody production projects. The completion of proteome-scale antibody libraries is expected to enable not only rapid verification of biomarkers, but also direct screening of antibodies by high-throughput assays. Here we report new methods that can screen antibodies on the basis of their reactivity with a large number of patient samples with reasonable comprehensiveness and throughput: automated quantification of immunofluorescence tissue microarray and immunofluorescence reverse-phase protein microarray (RPPM). Using these antibody-based proteomics technologies, we were able to identify biomarkers that can predict the efficacy of combinational chemotherapy, radiotherapy, and molecular targeting therapy. We also discuss the potential application of the antibody-based proteomics technologies to early-phase drug development.

Disease targeting therapies in patients with Eisenmenger syndrome: Response to treatment and long-term efficiency

ObjectivesTo examine long-term efficacy of disease targeting therapies (DTT) in patients with Eisenmenger syndrome. MethodsAll adult patients with Eisenmenger syndrome treated with DTT at our institution were included. Functional class (FC), oxygen saturation and 6-minute walk test distance (6MWTd) were analysed retrospectively. ResultsBetween 2002 and 2010, 79 Eisenmenger patients (21 males, 16 with Down syndrome) aged 34±10years (range 17–68years) were included. Median follow-up was 3.3years (range 0.2 to 8.9years). 6MWTd increased early after initiation of DTT, with a plateau after approximately 3years and no obvious trend towards a deterioration on average during longer-term follow-up. Two patients died during follow-up and escalation of treatment was required in 18 patients after a median period of 2.5years. Escalation of therapy was also associated with an increase in 6MWTd. In addition, FC improved on DTT and oxygen saturation, increased, both at rest and peak exercise. This effect was more pronounced in the patients with the lowest baseline oxygen saturation at rest. ConclusionsLong-term DTT is safe and improves objective exercise capacity and subjective symptoms. Response to DTT was typically observed early after initiation of DTT and was, on average, maintained longer-term. However, 1 in 5 patients required escalation of DTT, with time, due to symptomatic deterioration and this was associated with an afresh improvement in 6MWTd.

Flow Cytometry Detection of Intra-Cellular Tyrosine Kinase Inhibitors (TKI) Showed Variable Uptake in CML CD34+ Cells

Abstract 2747 IntroductionDespite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML), patient response is heterogeneous and it is generally accepted that residual disease and relapse are due to persistent CML cells, considered as leukemic stem cells. Their resistance has been related to lower TKI uptake. The amount of drug penetrating the targeted cells is most likely a major parameter of targeted therapy efficacy since it is essential that the therapeutic molecule be as close as possible to the target molecule. We developed a flow cytometry technique to analyze primary cells. MethodTo evaluate intracellular imatinib (ICIM) uptake, we developed a patented method based on natural UV fluorescence related to chemical structure. Consequently, since the difference in UV fluorescence units between treated and control cells is proportional to the amount of intra-cellular drugs, we validated this method after incubating K562 and KCL22 cell lines with TKI. The flow cytometry technique was standardized by using Flow-Check Fluorosphere calibrated beads immediately before, and at the end of, each series of analyses with a Coulter Epics Elite™ flow cytometer (Beckman Coulter) equipped with an Innova I90C-4 UV laser (Coherent). Then we analyzed primary blood cells from CML patients in chronic phase before any treatment. After lysis of erythrocytes, nucleated cells were incubated at 1.106 cells/ml with different doses of imatinib (IMA) (n=22), Nilotinib (NIL) (n=20) and Dasatinib (DAS) (n=20) at different times. Whenever possible, CML stem cells were analyzed using CD34-FITC staining. ResultsIn preliminary assays, we checked that there was a significant correlation between additional fluorescence measured by flow cytometry and the amount quantified by physico-chemical analysis after lysing a known number of cells (n=57, r2=0.73, p<0.001), which enabled us to convert UV fluorescence into pg of IMA per cell. Then we confirmed that IMI rapidly penetrated K562 and KCL22 cells (from 5 minutes of incubation) and reached a stable influx in viable cells from 1 hour (T1h). We chose this incubation time for further experiments. Similarly, we choose T2h for second generation TKI. We observed a dose-dependent accumulation in the two cells lines, but with differences at the lowest extra-cellular concentrations (1–5 μM) and not correlated with any membrane pump expression (OCT-1, ABCG2, ABCB1 and ABCC1). ICIM at T1h was correlated with cell sensitivity to IM at T24h expressed by the proportion of dead cells (r2=0.93 and 0.88 for K562 and KCL22 cells, respectively).We then applied our method to primary CML blood cells in comparison with normal blood cells. TKI penetrated all cell subsets, but amounts varied depending on cell sizes (FS/SS characteristics). The first data obtained with IM showed ICIM levels in CML cells that were relatively heterogeneous from one patient to another, ranging from 0.9 to 4 pg/cell for an extracellular concentration of 5 μM, i.e. a higher concentration (x 300) than in culture medium. The ability of the granulocyte cell lineage to store IMA was related to the Sokal prognostic index (p=0.05). We detected variable ICIM levels in CML CD34+ cells from 10/16 patients (0.04–0.7 pg/cell) and no signal for 6/16 patients. Surprisingly, the ability of CD34+ to store second generation TKIs is variable and not necessarily correlated to IMA uptake. DiscussionWe developed a simple, rapid flow cytometry method directly applicable to primary cells and requiring only few cells which makes it possible to identify target cell subsets, such as CML stem cells. The strong correlation between the ICIM amount and the sensitivity of CML cell lines to TKIs validated the method and suggested that ICIM could be a relevant biomarker for predicting the sensitivity of the CML clone. In our CML series, we observed striking inter-patient variability of the capacity of primary CML cells to store TKI. A correlation with the Sokal score suggests possible predictive value with regard to in vivo CML response to IMA, which could be taken into account when choosing TKI for first-line therapy. Furthermore, we observed marked heterogeneity between CML CD34+ cells for storing TKI that could partially explain the heterogeneity of in vivo response. The relationship between the ability of untreated CML CD34+ cells to store TKI and complete molecular response has to be established. Disclosures:No relevant conflicts of interest to declare.

Sanger method for detecting tissue EGFR in patients with non-small cell lung cancer

Objective To provide support for personalized targeted molecular therapy of lung cancer patients by exploring gene mutations of tissue epidermal growth factor receptor ( EGFR ) in nonsmall cell lung cancer.Methods Tissue samples were collected from 63 patients to extract DNA.Amplication of EGFR exons 18,19,20,and 21 was performed with PCR kit.The products of amplication were identified by dectrophoresis and then digested by enzymes.The products of digestion were amplified by PCR and then were purified.The end products were detected by the AB13130 genetic analyzer to obtain the wild-types or mutant status of EGFR exons 18,19,20,and 21.The relationship of EGFR gene mutations with the clinical pathophysiologieal characteristics and the efficacy of targeted therapy was analyzed.Results The mutation rate of EGFR exons 19 and 21 was 33.3％ ( 21/63 ),and the mutations were associated with the histological types and smoking status ( P＜ 0.05 ).The targeted therapy was beneficial for the patiens with EGFB mutation.Conclusions Sanger method for detecting gene mutations in NSCLC can provide technical support for the targeted therapy. Key words: Non-small cell lung cancer; Epidermal growth factor receptor; Gene mutation; Gene detection

Antiangiogenic therapies for malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM), arises from the mesothelial cells, is difficult to be diagnosed at an early stage, and is refractory to conventional chemotherapy and radiotherapy. Therefore, the establishment of novel effective therapies is necessary to improve the prognosis for many patients with this disease. Recent studies have demonstrated that angiogenesis plays a significant role in MPM progression, suggesting the importance of tumor vessels as therapeutic targets. To explore molecular pathogenesis and evaluate the efficacy of vascular targeting therapy in MPM, we developed orthotopic implantation SCID mouse models of MPM. We found that selective VEGF inhibitors were effective only in the treatment of high-VEGF-producing MPM models. On the other hand, multiple kinase inhibitor E7080, with inhibitory activity against various angiogenic cytokine receptors, suppressed the progression and prolonged survival of both high-VEGF-producing and low-VEGF-producing MPM models. Further understanding of the functional characteristics of tumor angiogenesis may be essential to improve targeting therapies in MPM. In this review, we introduce current status of clinical strategies and novel therapeutic approaches against angiogenesis in MPM.