A biomarker panel predicts recurrence-free survival in ulcerated primary cutaneous melanoma.

BACKGROUND: Ulceration is an independent adverse prognostic factor in cutaneous malignant melanoma (CMM). There is, however, a need for additional prognostic markers to identify patients with ulcerated stage I-II CMM who have a high-risk for recurrence. The aim of this study was to examine the prognostic impact of BRAF mutation, proliferation and presence of tumour infiltrating lymphocytes (TILs) in primary ulcerated CMM. MATERIAL AND METHODS: We have used a consecutive cohort consisting of 71 primary ulcerated CMM (T1b-T4b). BRAF mutation was detected using Cobas test and pyrosequencing. Protein expression of the proliferation marker Ki67 was analysed using immunohistochemistry. Presence of TILs was evaluated in representative hematoxylin-eosin stained formalin-fixed paraffin-embedded tumour sections. RESULTS: Proportion of BRAF mutated alleles, proliferation and presence of TILs all had a statistically significant impact on recurrence free survival in univariate analyses (HR 2.44, 95% CI 1.23-4.84, p = 0.011; HR 2.66, 95% CI 1.32-5.35, p = 0.006 respectively HR 0.48, 95% CI 0.24-0.98, p = 0.045). A trend test found a statistically significant decrease in the proportion of recurrence by including the three favourable factors (BRAF wildtype/low proportion of BRAF mutated alleles, low proliferation and high presence of TILs) (p = 0.0004). When at least two out of three factors were present there was a statistically significant association with longer recurrence free survival in the multivariate analysis (HR 0.30, 95% CI 0.15-0.61, p = 0.001) when adjusted for Breslow thickness, an established independent prognostic marker for CMM. CONCLUSION: Thus, this panel of markers could be an interesting novel concept for predicting the clinical outcome in patients with high-risk stage I-II ulcerated CMM.

miRNA-214 is related to invasiveness of human non-small cell lung cancer and directly regulates alpha protein kinase 2 expression.

The prognosis of non-small cell lung cancer (NSCLC) is poor, since it has often metastasized to distant organs by the time of diagnosis. Therefore, biomarkers predicting metastasis are crucial. miRNAs play important roles in the regulation of different tumor cell processes, including metastasis. We recently showed that miRNA-214 is linked to a radioresistant phenotype of NSCLC. miRNA-214 has been linked to metastasis in other tumor types. Therefore, we examined the role of miRNA-214 in the metastatic potential of NSCLC. We showed that downregulation of miRNA-214 increased invasive potential, and conversely, overexpression of miRNA-214 decreased invasiveness of NSCLC cells in vitro. Gene expression and bioinformatic analyses of NSCLC cells with ablated miRNA-214, identified a number of metastasis-related target genes, including pregnancy-associated plasma protein A (PAPP-A), alpha protein kinase 2 (ALPK2), cyclin-dependent kinase 6 (CDK6) and tumor necrosis-factor alpha-induced protein 3 (TNFAIP3). These were validated on mRNA and protein level to be regulated by miRNA-214. Through immunoprecipitation we showed that only ALPK2 is directly regulated by miRNA-214. We also examined the protein expression of these four genes in NSCLC tumors with respect to metastatic potential. These results showed that NSCLC tumors express these proteins at moderate-high levels in the nucleus, cytoplasm and/or plasma membrane although with no significant correlation to the overall survival or the metastatic potential of the patients. However, we also showed that the membrane-localized PAPP-A had a higher expression level compared to the cytoplasm-localized. In conclusion, we show that low miRNA-214 expression is linked to a higher invasive potential of NSCLC cells.

Quantitative proteomics profiling of primary lung adenocarcinoma tumors reveals functional perturbations in tumor metabolism.

In this study, we have analyzed human primary lung adenocarcinoma tumors using global mass spectrometry to elucidate the biological mechanisms behind relapse post surgery. In total, we identified over 3000 proteins with high confidence. Supervised multivariate analysis was used to select 132 proteins separating the prognostic groups. Based on in-depth bioinformatics analysis, we hypothesized that the tumors with poor prognosis had a higher glycolytic activity and HIF activation. By measuring the bioenergetic cellular index of the tumors, we could detect a higher dependency of glycolysis among the tumors with poor prognosis. Further, we could also detect an up-regulation of HIF1α mRNA expression in tumors with early relapse. Finally, we selected three proteins that were upregulated in the poor prognosis group (cathepsin D, ENO1, and VDAC1) to confirm that the proteins indeed originated from the tumor and not from a stromal or inflammatory component. Overall, these findings show how in-depth analysis of clinical material can lead to an increased understanding of the molecular mechanisms behind tumor progression.

Clinical activity of sorafenib in a previously treated advanced urothelial cancer patient.

A male patient, with advanced urothelial carcinoma, who had previously received cisplatin, was treated with sorafenib off-licence for 10.7 months. Evaluation of tumour response with computed tomography scans indicated a reduction in tumour size and necrosis of the metastases within 2 months. Progression-free survival was 10.5 months. Side effects were manageable and not beyond the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0 grade 2. Molecular profiling of two of the proposed targets of sorafenib, platelet-derived growth factor receptor ß and vascular endothelial growth factor receptor 2, of the patient's tumour lesion showed high and intermediate expression levels in the tumour as compared with the surrounding non-neoplastic tissue. In contrast to previous reports, we report a clinically meaningful effect of sorafenib in a patient with advanced urothelial carcinoma. Hence, it appears that a fraction of patients with this disease are sensitive to this compound. To identify subpopulations of responders, we propose that clinical trials evaluating sorafenib and other targeted drugs should be biomarker-driven and designed with endpoints that consider the mode of action of the specific compound.

Caspase-2 is a mediator of apoptotic signaling in response to gemtuzumab ozogamicin in acute myeloid leukemia.

The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg®) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.

Multiplex immune protein profiling of fine-needle aspirates from patients with non-small-cell lung cancer reveals signatures associated with PD-L1 expression and tumor stage.

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non-small-cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune-related proteins in fine-needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD-L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody-based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD-L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD-L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.

The prognostic implications of Notch1, Hes1, Ascl1, and DLL3 protein expression in SCLC patients receiving platinum-based chemotherapy.

OBJECTIVES: The aim was to analyse the tumor expression of Notch1, Hes1, Ascl1, and DLL3 in Small-Cell Lung Cancer (SCLC) and each such biomarker's potential association with clinical characteristics and prognosis after platinum-doublet chemotherapy (PDCT). MATERIAL AND METHODS: The protein expression of the biomarkers was evaluated using immunohistochemistry. Patients were categorized according to their sensitivity to first line PDCT: with a Progression-free survival (PFS) ≥ 3 months after completion of treatment considered "sensitive" and < 3 months after completion of treatment considered "refractory". PFS and overall survival were computed using Kaplan-Meier curves with 95% confidence interval. RESULTS AND CONCLUSION: The study included 46 patients, with 21 and 25 of the patients having "sensitive" and "refractory" disease, respectively. The majority of patients had a high DLL3 expression (n = 38), while a minority had Notch 1-high expression (n = 10). The chi-square test showed that there was a statistically significant negative association between Notch1 and Ascl1 expression (p = 0.013). The overall survival for patients with Notch1- high vs. low expression was 8.1 vs. 12.4 months, respectively (p = 0.036). Notch1 expression was an independent prognostic factor in the multivariate analysis (p = 0.02). No other biomarker showed any prognostic impact in this highly selected SCLC cohort. DLL3 is highly expressed in the majority of advanced staged SCLC cases, as expected. In the same patient population, Notch1 expression might have a potential prognostic implication, by driving a non-neuroendocrine differentiation process. Given the small number of cases with Notch1 high expression, the results of this study needs to be confirmed on a larger cohort.

Use of narrow-range peptide IEF to improve detection of lung adenocarcinoma markers in plasma and pleural effusion.

In this study we applied narrow-range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow-range IEF were run; IPG strips and free-flow electrophoresis. Data from this study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n=3) were compared with inflammatory pleuritis (n=3) using iTRAQ quantification. Using narrow-range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue-related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies.

The common Scandinavian human leucocyte antigen ancestral haplotype 62.1 as prognostic factor in patients with advanced malignant melanoma.

PURPOSE: We have previously demonstrated an association of the human leukocyte antigen (HLA), HLA-A2 allele with ovarian and prostate cancer mortality as well as a segregation of the ancestral HLA haplotype (AHH) 62.1 [(A2) B15 Cw3 DRB1*04] in patients with stage III-IV serous ovarian cancer. The objective of the present study was to determine the role of the HLA phenotype on the prognosis in stage III-IV malignant melanoma patients. PATIENTS AND METHODS: A cohort of metastatic malignant melanoma patients (n = 91), in stage III (n = 26) or IV (n = 65) were analysed for HLA-A, -B, -Cw and -DRB1 types by PCR/sequence-specific primer method. The frequencies of HLA alleles in the patients were compared to that of healthy Swedish bone marrow donors. The effect of HLA types on prognosis was defined by Kaplan-Meier and Cox analysis. RESULTS: The presence of the AHH 62.1 in clinical stage IV patients was significantly and independently associated with the worst survival rate recorded from the appearance of metastasis (HR = 2.14; CI = 1.02-4.4; P = 0.04). In contrast, the period from the primary diagnosis to metastasis was the longest in patients with this haplotype (HR = 0.40; CI = 0.17-0.90; P = 0.02). CONCLUSIONS: Melanoma patients in our cohort with 62.1 AHH which is associated with autoimmune diseases have an initial strong anti-tumour control with longer metastasis-free period. These patients have rapid progression after the appearance of metastasis, responding poorly to chemo- or/and immunotherapy. This apparently paradoxical clinical process could be due to the interplay between tumour clones escape and immune surveillance ending up with a rapid disease progression.

Tumor expression of S100A6 correlates with survival of patients with stage I non-small-cell lung cancer.

BACKGROUND: In a previously published in vitro study based on top-down proteomics we found that the calcium-binding proteins S100A6 and S100A4 were affected by exposure to ionizing radiation in a p53-dependent fashion. Both proteins showed post-translational modification changes, and S100A6 also showed increased expression and translocation in response to irradiation. Aim of the present study was to evaluate the expression of S100A6 and S100A4 in non-small-cell lung cancer (NSCLC). METHODS: S100A6 expression on archival tumor cell lysates from 39 patients with radically resected NSCLC was assessed with SELDI-TOF-MS. S100A6 identity was confirmed using a SELDI-based antibody-capture method on lysates from the A549 lung cancer cell line, cell lysates from two freshly prepared NSCLC samples, four plasma samples and one pleural effusion sample. Immunostainings for S100A6, S100A4 and p53 were performed on tissue microarrays containing 103 stage I surgically resected NSCLC cases and 14 normal lung parenchyma specimens. RESULTS: The presence of post-translationally modified S100A6 forms was confirmed with SELDI-MS on enriched tumor cell lysates, as well as in plasma and pleural effusion samples. In addition, high S100A6 peak intensity was associated with longer median survival (35 months vs. 18 months for high and low peak intensity, respectively; p=n.s.). The immunohistochemical analysis showed that 25% of tumors were S100A6 positive. S100A6 expression correlated directly with non-squamous histology (p<0.0001) and S100A4 expression (p=0.005), and inversely with p53 expression (p=0.01). S100A6-positive cases showed a trend of longer survival compared with S100A6-negative cases (p=0.07). This difference became significant when the analysis was restricted to p53-negative cases (n=72). In this subgroup of patients, whose tumors likely exhibit a functional p53, S100A6 was an independent prognostic factor of improved survival at multivariate analysis (HR 0.49, 95% CI 0.27-0.81, p=0.017). CONCLUSIONS: In this study we have validated on clinical material our previous findings on cell lines in terms of S100A6 expression and post-translational modifications pattern in NSCLC. Moreover, the survival results obtained in p53-negative stage I NSCLC cases support the proposed pro-apoptotic function of S100A6 and suggest the hypothesis of a cross regulation between these two proteins.

Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and for follow-up of personalized cancer therapy, including immunotherapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissue samples; however, the minute amounts of sample require sensitive multiplex molecular analysis to be of clinical biomarker utility. We have applied proximity extension assays (PEA) to analyze 167 proteins in FNA samples from patients with breast cancer (BC; n = 25) and benign lesions (n = 32). We demonstrate that the FNA BC samples could be divided into two main clusters, characterized by differences in expression levels of the estrogen receptor (ER) and the proliferation marker Ki67. This clustering corresponded to some extent to established BC subtypes. Our analysis also revealed several proteins whose expression levels differed between BC and benign lesions (e.g., CA9, GZMB, IL-6, VEGFA, CXCL11, PDL1, and PCD1), as well as several chemokines correlating with ER and Ki67 status (e.g., CCL4, CCL8, CCL20, CXCL8, CXCL9, and CXCL17). Finally, we also identified three signatures that could predict Ki67 status, ER status, and tumor grade, respectively, based on a small subset of proteins, which was dominated by chemokines. To our knowledge, expression profiles of CCL13 in benign lesions and BC have not previously been described but were shown herein to correlate with proliferation (P = 0.00095), suggesting a role in advanced BC. Given the broad functional range of the proteins analyzed, immune-related proteins were overrepresented among the observed alterations. Our pilot study supports the emerging role of chemokines in BC progression. Due to the minimally traumatic sampling and clinically important molecular information for therapeutic decisions, this methodology is promising for future immunoscoring and monitoring of treatment efficacy in BC.

Quantitative assessment of the subepithelial collagen band does not increase the accuracy of diagnosis of collagenous colitis.

The thickness of eosinophilic band in collagenous colitis (CC) was assessed by 3 methods: histologic estimates (22 observers), conventional measurements using a calibrated micrometric scale (1 observer), and semiautomatic micrometric measurements (1 observer). By the histologic estimate technique, 7.4% of the results failed to diagnose CC; by calibrated micrometry, the failure was 6% and by semiautomatic micrometry, 6%. The main difficulty in measuring the thickness of the CC band is that the deeper border of the band appears fuzzy and hairy-irregular. CC should be defined not exclusively on the basis of the thickness of the collagen table, but as a microscopic constellation characterized by a distorted superficial cell arrangement, with areas of epithelial denudation and inflammatory cells in the superficial epithelium and the lamina propria. In agreement with Lazenby's statement: "Focusing solely on the collagen band can result in both over- and underdiagnosis"

Exosomal RNA-profiling of pleural effusions identifies adenocarcinoma patients through elevated miR-200 and LCN2 expression.

HYPOTHESIS: The inherent challenges associated with tissue biopsies from lung have spurred an interest in the use of liquid biopsies. Pleural effusions are one source of liquid biopsy. Recently, extracellular vesicles of endocytic origin, exosomes, have attracted interest as liquid biopsy of tumors as they are thought to be a mirror of their tumor of origin. Here, we aimed to analyze if RNA profiling of exosomes isolated from pleural effusions could differentiate patients with lung adenocarcinoma from patients with benign inflammatory processes. METHODS: Exosomes were isolated from 36 pleural effusions from patients with adenocarcinoma (n = 18) and patients with benign inflammatory processes (n = 18). The two groups were balanced with respect to age and smoking history but with a gender bias towards males in the benign group. Profiling was conducted using RT-qPCR arrays covering 754 microRNAs and 624 mRNAs followed by statistical ranking of differentially regulated transcripts between the two patient cohorts. RESULTS: RNA profiling revealed differential expression of 17 microRNAs and 71 mRNAs in pleural effusions collected from patients with lung adenocarcinoma compared to pleural effusions from benign lung disease. Overall, top differentially expressed microRNAs, including miR-200 family microRNAs, provided a stronger diagnostic power compared to top differentially expressed mRNAs. However, the mRNA transcript encoding Lipocalin-2 (LCN2) displayed the strongest diagnostic power of all analyzed transcripts (AUC: 0.9916). CONCLUSIONS: Our study demonstrates that exosomal RNA profiling from pleural effusions can be used to identify patients with lung adenocarcinoma from individuals with benign processes and further proposes miR-200 microRNAs and LCN2 as diagnostic markers in lung cancer liquid biopsies.

Modulation of leukotriene signaling inhibiting cell growth in chronic myeloid leukemia.

Although tyrosine kinase inhibitors (TKIs) have dramatically improved clinical outcome in chronic myeloid leukemia (CML), cure rarely occurs. This may be due to BCR-ABL-independent, aberrant signaling pathways, one of which leads to leukotriene (LT) formation. Well-recognized as inflammatory mediators, LT can also affect oncogenic mechanisms of several tumors. We have previously discovered elevated LT-synthesis and up-regulated cysteinyl-LT-inducing enzyme in CML. Here we report on dose-dependent inhibition of CML cell growth exerted by specific blockers of LT-signaling. Thus, the cysteinyl-LT1-receptor-antagonist montelukast significantly reduced the growth of K562, KCL22, and KU812 cells, as well as primary CD34+ blood cells from two CML patients. Adding montelukast to the TKI imatinib caused combined inhibition. No effect was seen on normal bone marrow cells. Similarly, growth inhibition was also observed with the 5-lipoxygenase (LO)-inhibitor BWA4C, the 5-LO-activating-protein-(FLAP)-inhibitor licofelone and the LTB4(BLT1)-receptor-antagonist LY293111. Thus, blocking of aberrant LT-signaling may provide an additional, novel therapeutic possibility in CML.

Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells.

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.

Reliability of the reported size of removed colorectal polyps.

BACKGROUND: The size of colorectal polyps is important in the clinical management of these lesions. AIM: To audit the accuracy in calculating the size of "polyps" by various specialists. MATERIALS AND METHODS: Eighteen pathologists and four surgeons measured, with a conventional millimetre ruler, the largest diameter of 12 polyp phantoms. The results of two independent measurements (two weeks apart) were compared with the gold standard-size assessed at The Royal Institute of Technology, Sweden. RESULTS: Thirty-one percent (83/264-trial 1) and 33% (88/264-trial 2) of the measurements underestimated or overestimated the gold standard size by >1 mm. Of the 22 experienced participants, 95% (21/22-trial 1) and 91% (20/22-trial 2) misjudged by >1 mm the size of one or more polyps. Values given by 13 participants (4.9%) in trial 1 and by 15 participants (5.7%) in trial 2, differed by > or = +/-4 mm from the gold standard size. In addition, a big difference between the highest and the lowest values was recorded in some polyps (up to 11.4 mm). Those disparate values were regarded as a human error in reading the scale on the ruler. CONCLUSION: Using a conventional ruler (the tool of pathologists worldwide) unacceptably high intra-observer and inter-observer variations in assessing the size of polyp-phantoms was found. The volume and the shape of devices, as well as human error in reading the scale of the ruler were confounding factors in size assessment. In praxis, the size is crucial in the management of colorectal polyps. Considering the clinical implications of the results obtained, the possibility of developing a method that will allow assessment of the true size of removed clinical polyps is being explored.

Melphalan-flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma.

BACKGROUND: Chemotherapy options in advanced urothelial carcinoma (UC) remain limited. Here we evaluated the peptide-based alkylating agent melphalan-flufenamide (mel-flufen) for UC. METHODS: UC cell lines J82, RT4, TCCsup and 5637 were treated with mel-flufen, alone or combined with cisplatin, gemcitabine, dasatinib or bestatin. Cell viability (MTT assay), intracellular drug accumulation (liquid chromatography) apoptosis induction (apoptotic cell nuclei morphology, western blot analysis of PARP-1/caspase-9 cleavage and Bak/Bax activation) were evaluated. Kinome alterations were characterized by PathScan array and phospho-Src validated by western blotting. Aminopeptidase N (ANPEP) expression was evaluated in UC clinical specimens in relation to patient outcome. RESULTS: In J82, RT4, TCCsup and 5637 UC cells, mel-flufen amplified the intracellular loading of melphalan in part via aminopeptidase N (ANPEP), resulting in increased cytotoxicity compared to melphalan alone. Mel-flufen induced apoptosis seen as activation of Bak/Bax, cleavage of caspase-9/PARP-1 and induction of apoptotic cell nuclei morphology. Combining mel-flufen with cisplatin or gemcitabine in J82 cells resulted in additive cytotoxic effects and for gemcitabine also increased apoptosis induction. Profiling of mel-flufen-induced kinome alterations in J82 cells revealed that mel-flufen alone did not inhibit Src phosphorylation. Accordingly, the Src inhibitor dasatinib sensitized for mel-flufen cytotoxicity. Immunohistochemical analysis of the putative mel-flufen biomarker ANPEP demonstrated prominent expression levels in tumours from 82 of 83 cystectomy patients. Significantly longer median overall survival was found in patients with high ANPEP expression (P = 0.02). CONCLUSION: Mel-flufen alone or in combination with cisplatin, gemcitabine or Src inhibition holds promise as a novel treatment for UC.

Ephrin B3 interacts with multiple EphA receptors and drives migration and invasion in non-small cell lung cancer.

Ephrin receptors (Ephs) are reported to control metastatic signaling of non-small cell lung cancer (NSCLC) and other tumors. Here we show for the first time that blocking expression of the Eph ligand Ephrin B3 inhibits NSCLC cell migration and invasion. We demonstrate that Ephrin B3 directly binds the EphAs EphA2, EphA3, EphA4, and EphA5. EphA2 Ser897 was previously shown to drive migration propensity of tumor cells and our study reveals that EphA2 stays phosphorylated on Ser897 in the Ephrin B3/EphA2 complex in NSCLC cells of different histology. Moreover, we report that within such Ephrin B3/EphA2 complex both Akt Ser 129 and p38MAPK are found indicating a potential to drive migration/proliferation. We also found the EMT marker E-cadherin expression to be maintained or increased upon Ephrin B3 blockade in NSCLC cells. Expression of Ephrin B3 was furthermore analyzed in a cohort of NSCLC stage IA-IB cases (n=200) alongside EphA2 and Ephrin A1. We found that Ephrin B3 was concomitantly expressed with EphA2 and Ephrin A1 with higher Ephrin B3 levels found in non-squamous than in squamous tumors, whereas EphA2 was higher expressed in well-differentiated than in low-differentiated tumors. In the entire NSCLC cohort, Ephrin B3 expression was not linked to patient survival, whereas a high EphA2 expression was associated with improved survival (p=0.03). In conclusion, we show that blocking Ephrin B3 expression inhibits NSCLC proliferation-, migration- and invasion capacity which calls for further studies on interference with Ephrin B3 as a possible therapeutic avenue in this tumor malignancy.

Modulation of response to radiation of human lung cancer cells following insulin-like growth factor 1 receptor inactivation.

Targeted disruption of the insulin-like growth factor 1 receptor (IGF-1R) restricts proliferation of tumor cells and enhances their in vitro radiosensitivity. However, there is little information regarding the effect of IGF-1R expression and function on the lung cancer response to radiotherapy. In this study, we evaluated the cell surface expression of IGF-1R and the antitumoral effect of IGF-1R blockade in combination with irradiation in 6 non-small cell lung cancer (NSCLC) cell lines. All cell lines showed specific IGF-1 binding with an affinity ranging from 0.95x10(-9) to 2.3x10(-9) M, which was evaluated by competitive binding assay. The amount of binding sites ranged from 118 to 377 fmol/mg protein. In one cell line (U1810), the combined treatment led to synergistic cell death and was associated with an accumulation of cells in the G2 phase. IGF-1R activation was able to obstruct serum starvation/radiation-induced cell death in U1810 cell line. Additive interactions were found for four cell lines (A549, H157, H23 and H125) whereas only subadditive effects were observed in U1752 cell line. Our results indicate that the IGF-1R is present on NSCLC cells and thereby its involvement in the modulation of radiosensitivity in lung cancer cells.

NRAS and BRAF mutations arise early during melanoma pathogenesis and are preserved throughout tumor progression.

PURPOSE: Recently, it was reported that BRAF mutations are frequent in melanoma. Previously, we analyzed a large series of paired primary and metastatic melanomas for NRAS codon 61 mutations and showed that they arise early and are preserved during tumor progression. Here, we have screened the same tumor samples for BRAF mutations. EXPERIMENTAL DESIGN: Primary melanomas (n = 71) and corresponding metastases (n = 88) from 71 patients were screened for BRAF exon 11 and exon 15 mutations using single-strand conformational polymorphism and nucleotide sequence analysis RESULTS: BRAF mutations were found in 42 of 71 patients (59%). Thirty-seven patients had mutations that lead to a Val599Glu change, whereas mutations resulting in Gly468Ser, Val599Arg, Val599Lys, and Lys600Glu changes were detected in one patient each. Furthermore, one patient had a 6-bp insertion between codons 598 and 599, encoding two threonine residues. In most cases, paired primary and metastatic lesions had the same BRAF genotype (i.e., mutations present in the primary tumors were preserved in the corresponding metastases, and mutations did not arise at the metastatic stage if they were not present in the primary lesion). Using laser-capture microdissection, BRAF mutations were found in the radial growth phase of the primary lesions. BRAF mutations occurred exclusively in tumors that were wild type for NRAS, and in total, 89% of the patients analyzed (63 of 71) had mutations in either of these two genes. CONCLUSIONS: The Ras-Raf-mitogen-activated protein kinase/extracellular signal-regulated kinase-extracellular signal-regulated kinase signaling pathway is activated in the vast majority of melanomas. Activation occurs through either NRAS or BRAF mutations, both of which arise early during melanoma pathogenesis and are preserved throughout tumor progression.