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Supplementary Figure 1 from KIT Pathway Alterations in Mucosal Melanomas of the Vulva and Other Sites

Purpose: A significant proportion of mucosal melanomas contain alterations in KIT. The aim of this study was to characterize the pattern of KIT, NRAS, and BRAF mutations in mucosal melanomas at specific sites and to assess activation of the KIT downstream RAF/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in mucosal melanoma specimens.Experimental Design: Seventy-one primary mucosal melanomas from various sites were studied. Mutation analysis was done by DNA sequencing. Expression of KIT, phosphorylated (p)-ERK, and p-AKT was evaluated by immunohistochemistry.Results: KIT mutations were detected in 35% (8 of 23) of vulvar, 9% (2 of 22) of anorectal, 7% (1 of 14) of nasal cavity, and 20% (1 of 5) of penile melanomas. No KIT mutations were found in 7 vaginal melanomas. The difference in KIT mutation frequency between vulvar and nonvulvar cases was statistically significant (P = 0.014). The overall frequencies of NRAS and BRAF mutations were 10% and 6%, respectively. Notably, vaginal melanomas showed a NRAS mutation rate of 43%. KIT gene amplification (≥4 copies), as assessed by quantitative real-time PCR, was observed in 19% of cases. KIT expression was associated with KIT mutation status (P < 0.001) and was more common in vulvar than nonvulvar tumors (P = 0.016). Expression of p-ERK and p-AKT was observed in 42% and 59% of tumors, respectively, and occurred irrespective of KIT/NRAS/BRAF mutation status. NRAS mutation was associated with worse overall survival in univariate analysis.Conclusions: Results show that KIT mutations are more common in vulvar melanomas than other types of mucosal melanomas and that both the RAF/MEK/ERK and PI3K/AKT pathways are activated in mucosal melanoma specimens. Clin Cancer Res; 17(12); 3933–42. ©2011 AACR.

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. 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. 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. 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.

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.

ObjectivesThe 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 methodsThe 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 conclusionThe 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.

// Ghazal Efazat 1, * , Metka Novak 1, * , Vitaliy O. Kaminskyy 2 , Luigi De Petris 1 , Lena Kanter 1 , Therese Juntti 1 , Per Bergman 3 , Boris Zhivotovsky 2 , Rolf Lewensohn 1 , Petra Haag 1 , Kristina Viktorsson 1 1 Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, SE-171 76 Stockholm, Sweden 2 Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, SE-171 77 Stockholm, Sweden 3 Department of Molecular Medicine and Surgery (MMK), Thoracic Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden * These authors have contributed equally to this work Correspondence to: Kristina Viktorsson, email: Kristina.Viktorsson@ki.se Petra Haag, email: Petra.Haag@ki.se Keywords: Ephrin B3, EphA2, NSCLC, migration, invasion Received: September 17, 2015 Accepted: July 16, 2016 Published: August 11, 2016 ABSTRACT 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.

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.

Summary Background The prognostic impact of several histopathological prognostic features in cutaneous malignant melanoma (CMM) remains controversial. Objectives To assess the independent prognostic value of mitotic rate, regression, tumour-infiltrating lymphocytes (TILs) and growth phase in primary stage I and II CMMs. Methods Clinicohistopathological data were obtained from the Stockholm–Gotland registry for 4237 patients diagnosed with an incident primary stage I or II CMM followed up to December 2011. The risk of CMM-specific death was evaluated by a Cox regression model. Results A mitotic rate of 1–10 mitoses per mm2 [hazard ratio (HR) 1·69, 95% confidence interval (CI) 1·16–2·45] and > 10 mitoses per mm2 (HR 2·27, 95% CI 1·46–3·52) were significant; TILs and regression were not. A more detailed analysis of data assessed between 1989 and 1995 confirmed significantly increased HRs for the presence vs. absence of mitoses (HR1–5/mm² 2·25, 95% CI 1·36–3·76; HR6–10/mm² 2·34, 95% CI 1·23–4·44; HR> 10/mm² 2·64, 95% CI 1·39–4·99). Other prognosticators were increasing T-stage vs. T1, presence of ulceration and presence of vertical growth phase (VGP). In T1 CMMs, an increasing tumour thickness vs. 0·8 mm 2·92, 95% CI 1·57–5·43) and presence of ulceration were significantly associated with higher HRs; mitotic rate, TILs, regression and growth phase were not. Conclusions Determinants of increased risk of CMM death in stage I and II CMMs were increasing T-stage, presence of ulceration, presence of mitoses and VGP. This was not found for TILs or regression.

A major challenge in muscle-invasive urothelial carcinoma (UC) is to identify biomarkers that can predict disease prognosis and treatment response after cystectomy. Therefore, we analyzed the potential prognostic value of the proteins vascular endothelial growth factor receptor 2 (VEGFR2), S100A4, and S100A6 in UC.Retrospective outcome data and tumor specimens from 83 cystectomy patients with histologically confirmed invasive UC were included. Expression levels of VEGFR2 (also called flk-1 and KDR), S100A4, and S100A6 were analyzed in primary tumor tissue by immunohistochemistry.Immunohistochemical staining and analysis of VEGFR2, S100A4, and S100A6 showed localization mainly in tumor cell cytoplasm. High VEGFR2 expression and low tumor category were independent variables associated with longer overall survival (OS) and disease-free survival, revealed by a bivariate Cox proportional hazards regression model (both P0.001). In addition, the univariate log-rank test and the Cox model demonstrated that OS beyond 2 years was significantly greater among patients with low S100A6 expression than in those with high S100A6 expression (P = 0.017 and 0.022, respectively). Differences in tumor expression of S100A4 were not significantly associated with outcome.In this study, VEGFR2 expression was significantly correlated with risk of disease relapse and OS in a defined cohort of patients with UC of the bladder treated by cystectomy.

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.

Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem‑cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell‑induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non‑random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

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 β 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.

Clinical management of primary cutaneous melanomas is based on histopathological staging of the tumour. The aim of this study was to investigate, in a non-selected population in clinical practice, the agreement rate between general pathologists and pathologists experienced in melanoma in terms of the evaluation of histopathological prognostic parameters in cutaneous malignant melanomas, and to what extent the putative variability affected clinical management. A total of 234 cases of invasive cutaneous malignant melanoma were included in the study from the Stockholm-Gotland Healthcare Region in Sweden. Overall interobserver variability between a general pathologist and an expert review was 68.8-84.8%. Approximately 15.5% of melanomas 1 mm after review. In conclusion, review by a pathologist experienced in melanoma resulted in a change in recommendations about surgical excision margins and/or sentinel node biopsy in subgroups of Ti melanomas.

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

Background: Resistance to endocrine therapy is a clinical problem and is not explained by lack or loss of the oestrogen receptor (ER). Up-regulation of several receptor tyrosine kinases have been correlated to diminished effect of endocrine treatment. More contradictory are the role of downstream mitogen activated protein kinases (MAPK's). Patients and Methods: We performed a tissue micro array (TMA's) on patients that originate from an earlier presented population of 404 patients with a steroid receptor positive BC subjected to tamoxifen as only adjuvant systemic treatment. Material from 120 patients was available for the TMA. JNK, p38 and ERK was determined by use of immunohistochemistry and scored 0, +1, +2, +3. Patients were grouped into two categories for statistical analyses. Results: Absence or low JNK was statistically significantly correlated with HER2+ (p=0.018), a Luminal B like subtype (ER+, high proliferation and/or HER2+) (p=0.007), shorter relapse-free (p= 0.0494) and breast cancer corrected survival (p=0.0458). High ERK was more frequent among node-negative patients (p=0.00469) and more frequent in relapse-free patients; RFS (p= 0.225); BCCS (p= 0.0705). High expression of p38 was associated with p38 previously determined by ELISA (p=0.085) and HER2+ (p=0.094), however not reaching statistical significance. High p38 was related to increased relapses within the first years but the difference vanished with longer follow-up time; RFS (p=0.486) and BCCS (p= 0.589). JNK remained as an independent marker of RFS in a Cox proportional multivariate analysis (HR=2.19; 95%CI=1.14−11.41; p=0.029), together with nodal status (HR=3.6; 95%CI=1.44−8.81; p=0.006) and histological grade (HR=3.1; 95%CI=1.29−7.56; p=0.011). Discussion: Our data on ERK and p38 is concordant with results from previous published studies. Two studies have shown ERK expression, determined by IHC to be correlated with “good prognosis features” as smaller tumour size, absence of nodal metastasis, lower proliferation and a favourable outcome after adjuvant tamoxifen (Svensson et. al. 2005, Bergqvist et. al. 2006). Higher p38 determined by use of an ELISA has been suggested as a marker of early relapses i.e. intrinsic resistance, although methodological problems must be taken into account (Linderholm et. al. 2011). By use of IHC we could confirm that patients with higher expression of p38 actually had significantly more relapses within early follow-up. JNK was the MAPK with the most pronounced effect on survival. Absent or low expression was significantly correlated with an impaired survival in both uni- and multivariate analyses, a finding to our very best knowledge not described before. Low JNK was actually found more frequently in patients with a HER2 positive or Luminal B like BC (ER+, high proliferation rate or HER2+). Conclusions: Our results suggest that MAPK's can be evaluated by IHC on TMA's. Absence/low JNK was significantly correlated with an impaired survival and a Luminal B like subtype. Confirming studies in large preferable randomised patient populations are warranted. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-19.

Background: Radiotherapy is central in the treatment of cervical cancer. The formation of DNA double-strand breaks is considered to be critical for the radiotherapeutic effect. The non-homologous end joining (NHEJ) proteins DNA–PKcs, Ku70 and Ku86 have a major role in repairing DNA lesions. The objective of this study was to analyse if the expression of DNA–PKcs, Ku70 and Ku86 and their downstream signalling molecules p53, p21 and Mdm-2 are altered in residual cervical tumours after radiotherapy. Methods: Retrospective analysis of 127 patients with cervical cancer stage IB-IIA treated with preoperative radiotherapy and radical surgery, revealed residual tumour in the cervical specimen in 30 patients. In 22 cases tumour material from residual and corresponding primary tumour were retrieved and the expression of DNA–PKcs, Ku86, Ku70, p53, p21 and Mdm-2 were assessed by immunohistochemistry. Results: Residual tumours showed increased frequency of DNA–PKcs (P=0.037), Ku70 (P=0.018), Ku86 (P=0.008) positive cells. A correlation in DNA–PKcs expression between primary and residual tumours was found. The frequency of p21-positive cells was decreased (P=0.007) in residual tumours whereas no change in p53 or Mdm-2-positive cells were observed. Conclusion: Our results show that cervical carcinoma surviving radiotherapy have an increased DNA–PK expression. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of DNA–PK function may be part of a radioresistance mechanism within this tumour type.

For clinically relevant studies on melanoma progression and invasiveness, in vivo experimental systems with a human cellular microenvironment would be advantageous. We have compared tumor formation from a human cutaneous malignant melanoma cell line (BL), after injection as conventional xenografts in the mouse, or when injected into a predominantly species-specific environment of human embryonic stem cell–derived teratoma induced in the mouse (the hEST model). The resulting melanoma histology was generally analogous, both systems showing delimited densely packed areas with pleomorphic cells of malignant appearance. A specificity of the integration process into the human embryonic teratoma tissues was indicated by the melanoma exclusively being found in areas compatible with condensed mesenchyme, similar to neural crest development. Here, also enhanced neovascularization was seen within the human mesenchymal tissues facing the BL melanoma growth. Furthermore, in the hEST model an additional melanoma cell phenotype occurred, located at the border of, or infiltrating into, the surrounding human loose mesenchymal fibrous stroma. This BL population had a desmoplastic spindle-like appearance, with markers indicative of dedifferentiation and migration. The appearance of this apparently more aggressive phenotype, as well as the induction of human angiogenesis, shows specific interactions with the human embryonic microenvironment in the hEST model. In conclusion, these data provide exciting options for using the hEST model in molecular in vivo studies on differentiation, invasiveness, and malignancy of human melanoma, while analyzing species-specific reactions in vivo. [Cancer Res 2009;69(9):3746–54]

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 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.

Objective Gemtuzumab ozogamicin (GO), comprising a CD33 antibody linked to the toxin calicheamicin, represents a novel and promising targeted therapy in acute myeloid leukemia (AML). The more definite mechanisms by which GO exerts its cell death−inducing propensity, and thus how sensitivity and resistance to GO are regulated, still remain to be elucidated. We have studied proapoptotic signaling events induced by GO and free calicheamicin in AML cells. Materials and Methods AML cell lines and primary blood cells from six patients with acute leukemia were incubated with GO or calicheamicin and the effects on cell viability and proapoptotic signaling were analyzed using MTT assay, flow cytometry, immunofluorescence and immunoblotting. Results GO and free calicheamicin at clinically relevant concentrations resulted in decreased cell viability, appearance of apoptotic morphology, depolarization of mitochondria, and activation of caspase-3 signaling in HL60 and NB4 AML cells. In contrast, none of these events were observed in GO-exposed KG1a AML cells. Notably, GO treatment also caused proapoptotic conformation of Bak and Bax and activation of stress-activated protein kinase p38 in responsive but not in resistant AML cells. In patient-derived AML cells, GO and calicheamicin induced a heterogeneous cytotoxic response, partly linked to CD33 expression and with signs of caspase-3 activation. Conclusion Our novel data on GO-induced proapoptotic activation of Bax, Bak, and stress-activated protein kinase indicate an important role for these signal proteins in the regulation of GO sensitivity in AML.

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.”

Our goal in this paper is to show an analytical workflow for selecting protein biomarker candidates from SELDI-MS data. The clinical question at issue is to enable prediction of the complete remission (CR) duration for acute myeloid leukemia (AML) patients. This would facilitate disease prognosis and make individual therapy possible. SELDI-mass spectrometry proteomics analyses were performed on blast cell samples collected from AML patients pre-chemotherapy. Although the biobank available included approximately 200 samples, only 58 were available for analysis. The presented workflow includes sample selection, experimental optimization, repeatability estimation, data preprocessing, data fusion, and feature selection. Specific difficulties have been the small number of samples and the skew distribution of the CR duration among the patients. Further, we had to deal with both noisy SELDI-MS data and a diverse patient cohort. This has been handled by sample selection and several methods for data preprocessing and feature detection in the analysis workflow. Four conceptually different methods for peak detection and alignment were considered, as well as two diverse methods for feature selection. The peak detection and alignment methods included the recently developed annotated regions of significance (ARS) method, the SELDI-MS software Ciphergen Express which was regarded as the standard method, segment-wise spectral alignment by a genetic algorithm (PAGA) followed by binning, and, finally, binning of raw data. In the feature selection, the "standard" Mann-Whitney t test was compared with a hierarchical orthogonal partial least-squares (O-PLS) analysis approach. The combined information from all these analyses gave a collection of 21 protein peaks. These were regarded as the most potential and robust biomarker candidates since they were picked out as significant features in several of the models. The chosen peaks will now be our first choice for the continuing work on protein identification and biological validation. The identification will be performed by chromatographic purification and MALDI MS/MS. Thus, we have shown that the use of several data handling methods can improve a protein profiling workflow from experimental optimization to a predictive model. The framework of this methodology should be seen as general and could be used with other one-dimensional spectral omics data than SELDI MS including an adequate number of samples.

In solid tumors, human leucocyte antigen (HLA)-A2 has been suggested to be a risk factor and a negative prognostic factor. The HLA-A2 allele in Scandinavia has a high prevalence; it decreases with latitude and also with ovarian cancer mortality in Europe. Furthermore, an association of the HLA-A2 allele with severe prognosis in serous adenocarcinoma of the ovary in stages III-IV was found. Thirty-two unrelated Swedish women with relapsing or progressive ovarian cancer were analysed for the genotypes at the HLA-A, HLA-B, HLA-Cw, and HLA-DRB1 loci by the polymerase chain reaction/sequence-specific primer method. The frequencies of HLA alleles of healthy Swedish bone marrow donors provided by the coordinating centre of the Bone Marrow Donors Worldwide Registries, Leiden, the Netherlands were used as controls. When this cohort of epithelial ovarian cancer patients was compared with healthy Swedish donors, the frequency of HLA-A1 and HLA-A2 gene/phenotype appears, although not statistically significant, to be increased in patients with ovarian carcinoma, while HLA-A3 was decreased. HLA-A2 homozygotes were twofold higher in patients. The A2-B8 haplotype was significantly increased (corrected P value). A2-B5, A2-B15, A2-DRB1*03, A2-DRB1*04, A2-B15-Cw3, and A2-B8-DRB1*03 had odds ratio as well as the level of the lower confidence interval above 1 and significant P value only when considered as single, non-corrected analysis. HLA-B15 and HLA-Cw3 were only present in HLA-A2-positive patients showing that the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes were segregated. In this selected cohort with advanced disease, there are indications of an unusual overrepresentation of HLA class I and II genes/haplotypes as well as segregation for the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes. These findings are presented as a descriptive analysis and need further investigations on a larger series of ovarian cancer patients to establish prognostic associations.

The insulin-like growth factor-1 receptor (IGF-1R) mitogenic signaling mediates malignant cell survival by many complex and redundant pathways. This study compared the effects of IGF-1R inhibition on viability and apoptosis of two NSCLC cell lines, using three different methods for the impairment of IGF-1R function: (IR3, an anti-IGF-1R antibody; tyrphostin AG1024, a tyrosine kinase inhibitor (TKI) and IGF-1R-small interfering RNA (siRNA). IGF-1R inhibition led to a decrease of cell survival and induced apoptosis in a manner depending on the approach used for the receptor inhibition. To find an explanation, we analyzed the effects of these treatments on three major antiapoptotic pathways evoked by IGF-1R signaling: IRS-1, Shc and 14.3.3-dependent mitochondrial translocation of Raf-1 kinase (mitRaf). (IR3 downregulated IRS-1 phosphorylation in A549 cells and Shc phosphorylation in U1810 cells. While in A549 cells AG1024 treatment decreased both IRS-1 and Shc phosphorylation, in U1810 cells the IRS-1 phosphorylation was only slightly affected and the Shc phosphorylation drastically downregulated. Neither (IR3 nor AG1024 had any effect on Raf-1 kinase translocation. Irrespective of the cell line, IGF-1R-siRNA treatment induced downregulation of both IRS-1 and Shc phosphorylation coupled with the abrogation of mitRaf. In addition, the IGF-1R-siRNA proved to be the most potent inducer of apoptosis suggesting that more than one antiapoptotic pathway in IGF-1R signaling should be inhibited to effectively induce apoptosis in lung cancer cells.

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.

The primary aim of this study was to investigate if the expression of the DNA damage identifying protein DNA-PKcs known to be involved in DNA repair after treatment with ionising radiation can be used as a predictive marker for radiotherapy (RT) response in cervical cancer. Formalin-fixed primary tumour biopsies from 109 patients with cervical cancer, FIGO-stage IB–IIA, treated with preoperative brachytherapy followed by radical surgery were analysed by immunohistochemistry. In addition, correlation studies between early pathological tumour response to radiation and expression of Ku86, Ku70, Mdm-2, p53 and p21 in primary tumours were also performed. We found that tumour-transformed tissue shows positive immunostaining of DNA-PKcs, Ku86 and Ku70, while non-neoplastic squamous epithelium and tumour-free cervix glands show negative immunoreactivity. Expression of DNA-PKcs positively correlated with both Ku86 and Ku70, and a statistically significant correlation between the Ku subunits was also found. After RT, 85 patients demonstrated pathologic complete remission (pCR), whereas 24 patients had residual tumour in the surgical specimen (non-pCR). The main finding of our study is that there was no correlation between the outcome of RT and the expression of DNA-PK subunits. Positive p53 tumours were significantly more common among non-pCR cases than in patients with pCR (P=0.031). Expression of p21 and Mdm-2 did not correlate with the outcome of RT.

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 0.95×10 −9 to 2.3×10 −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 G 2 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.

We have published previously a prototype of a decision model for anaemic patients with myelodysplastic syndromes (MDS), in which transfusion need and serum erythropoietin (S-Epo) were used to define three groups with different probabilities of erythroid response to treatment with granulocyte colony-stimulating factor (G-CSF) + Epo. S-Epo 500 U/l and >/= 2 units/month for a poor response, whereas the presence of only one negative prognostic marker predicted an intermediate response. A total of 53 patients from a prospective study were included in our evaluation sample. Patients with good or intermediate probability of response were treated with G-CSF + Epo. The overall response rate was 42% with 28.3% achieving a complete and 13.2% a partial response to treatment. The response rates were 61% and 14% in the good and intermediate predictive groups respectively. The model retained a significant predictive value in the evaluation sample (P < 0.001). Median duration of response was 23 months. Scores for global health and quality of life (QOL) were significantly lower in MDS patients than in a reference population, and fatigue and dyspnoea was significantly more prominent. Global QOL improved in patients responding to treatment (P = 0.01). The validated decision model defined a subgroup of patients with a response rate of 61% (95% confidence interval 48-74%) to treatment with G-CSF + Epo. The majority of these patients have shown complete and durable responses.

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.

Insulin-like growth factor-1 receptor (IGF-1R) has been shown to be important for melanoma cell growth and survival. In this study we first show, using immunohistochemistry, that progression from benign nevi to malignant melanoma is paralleled by an increased expression of IGF-1R and a down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. Even though the expression of p27Kip1 was drastically reduced compared to benign tumors, detectable amounts of it could be assayed by Western blotting in cultured melanoma cells. To analyze whether there is a causative relationship between the IGF-1 pathway and p27Kip1 expression, melanoma cells were treated with alpha IR-3, an antibody blocking the IGF-1 binding to IGF-1R, or Tunicamycin, which inhibits the translocation of IGF-1R to the cell surface. From these studies we could conclude that the overall expression of p27Kip1 is independent of the IGF-1 pathway. In contrast, the association of p27Kip1 with the different cyclins was drastically affected. Both TM and alpha IR-3 decreased the binding of p27Kip1 to cyclin D1, whose expression was drastically reduced. On the other hand there was an increased binding of p27Kip1 to cyclin E and cyclin A. This redistribution of p27Kip1 may be a mechanism for growth arrest and induction of apoptosis following interruption of the IGF-1 pathway in melanoma cells.

Background In an epidemiologic study of 219 Swedish females with vulvar melanoma, the authors previously established the incidence of this disease as 0.19 per 100,000 women, with a 3% annual decrease from 1960 to 1984 and a 5-year relative survival rate of 47%. After reviewing the medical histories of all of the 219 patients, the authors documented their precise clinical and histopathologic features, which, along with treatment, are assessed herein as predictors of survival. Methods Of 219 consecutive cases of vulvar melanoma collected from the Swedish National Cancer Registry, 21 were excluded because of inadequate data. Clinical and histopathologic materials from the remaining 198 cases were then reexamined. With a clinical three-stage system, lesion types and treatment modalities were assessed as survival factors in univariate and multivariate analyses. Results In univariate analysis, significant predictors of survival for patients at Stage I were tumor thickness, ulceration, number of mitoses, macroscopic amelanosis, preexisting nevi, extent of tumor invasion (lateral labia majora), and patient age. The mode of treatment was not significant. In multivariate analysis, staging (Stage I vs. II and III) and tumor thickness were independent predictors of survival. For Stage I only, tumor thickness, ulceration, and clinical amelanosis independently predicted survival time. Conclusions To the authors' knowledge, this is the largest series of patients with vulvar melanoma ever reviewed, and an ethnically homogeneous and nationwide female population is represented. In this series, clinical stage, macroscopic amelanosis, and tumor characteristics such as tumor thickness and ulceration, rather than treatment mode, were the best factors for predicting the outcome of these patients.

BACKGROUND Because the clinical and histopathologic features of vulvar melanoma had not been characterized completely in a large, homogeneous population, the authors retrospectively analyzed all such patients recorded in Sweden during a 25-year period. METHODS The Swedish National Cancer Registry opened its records to the authors for review of all 219 females with primary vulvar melanoma reported from 1960 to 1984. Histopathologic specimens and clinical histories of the 198 patients who qualified for this study were reanalyzed and the tumors rigorously subtyped. RESULTS Macroscopically amelanotic tumors were observed in 27% of patients, predominantly in glabrous skin; the clitoral area and labia majora were the most common primary sites. Of all melanomas, 46% emerged in glabrous skin, 12% emerged in hairy skin, and 35% extended to both areas. On average, approximately 2.5 times more melanomas appeared in the vulva than on the whole body surface. Overall, 57% were of the mucosal lentiginous (MLM) type, 22% were nodular melanomas (NMs), 12% were unclassified, and only 4% were superficial spreading melanomas (SSMs); this was the reverse of the order observed for cutaneous melanoma. Almost all vulvar melanomas underwent a vertical growth phase; other common features were marked thickness and ulceration, particularly in the glabrous skin. Preexisting nevi occurred in 11 cases, all in hairy skin, and 71% in conjunction with SSM but only 4% with MLM. CONCLUSIONS Several clinical and histopathologic features indicated that the natural history of vulvar melanomas is at variance with that of cutaneous melanomas. Because preexisting nevi, which are often considered a precursor to melanoma, were significantly linked to SSM and only in the vulvar hairy skin, melanomas in the glabrous skin apparently emerged de novo. Cancer 1999;86:1273–84. © 1999 American Cancer Society.

Thrombocytopenia is a substantial clinical problem for patients with myelodysplastic syndromes (MDS). Cytokine treatment for granulocytopenia and anaemia may further reduce the platelet counts. We studied serum thrombopoietin levels (S-TPO) in 52 patients with MDS and 96 healthy controls and related the results to clinical and morphological variables. S-TPO was also assessed after treatment with granulocyte-CSF (G-CSF) and erythropoietin (EPO) in 30 of these patients. S-TPO in MDS was not a normally distributed variable; mean value was 394 pg/ml, SD ±831 and median value 123 (12–5000 pg/ml). The controls showed lower S-TPO levels than the patients (median 78 pg/ml, P = 0.003) whereas no differences between the MDS subgroups were observed (P = 0.86). Patients with ringed sideroblastic anaemia (RARS) showed the highest platelet counts and higher S-TPO levels than the controls (P = 0.005). No association between platelet counts and S-TPO levels was found in the patients (P = 0.67). TPO levels were generally low in patients with refractory anaemia with an excess of blasts (RAEB), but very high levels were found in five patients. Patients with a high transfusion need had higher S-TPO levels, whereas bone marrow blast counts, cellularity or megakaryocytes showed no correlation with S-TPO. Patients with 5q− showed lower TPO levels than the other patients, indicating that thrombopoietin is not a mediator of thrombocytosis in these cases. Treatment with G-CSF + EPO significantly reduced the platelet counts (P = 0.0002), but this change was not related to significant changes in S-TPO levels or morphology. Patients with RARS and thrombocytosis who normalized their platelet counts showed a concomitant reduction in S-TPO. This may suggest that the increased platelet counts observed in RARS may be caused by increased S-TPO levels. In conclusion, our study shows that platelet, megakaryocyte and thrombopoietin regulation is rather complex in myelodysplastic syndromes and that spontaneous or induced thrombocytopenia are not usually mirrored by increased S-TPO levels.

In this study we provide evidence that the low expression of IGF-1R at the cell surface of estrogen-independent breast cancer cells is due to a low rate of de novo synthesis of dolichyl phosphate. The analyses were performed on the estrogen receptor-negative breast cancer cell line MDA231 and, in comparison, the melanoma cell line SK-MEL-2, which expresses a high number of plasma membrane-bound IGF-1R. Whereas the MDA231 cells had little or no surface expression of IGF-1R, they expressed functional (i.e., ligand-binding) intracellular receptors. By measuring the incorporation of [3H]mevalonate into dolichyl phosphate, we could demonstrate that the rate of dolichyl phosphate synthesis was considerably lower in MDA231 cells than in SK-MEL-2 cells. Furthermore, N-linked glycosylation of the alpha-subunit of IGF-1R was 8-fold higher in the melanoma cells. Following addition of dolichyl phosphate to MDA231 cells, N-linked glycosylation of IGF-1R was drastically increased, which in turn was correlated to a substantial translocation of IGF-1R to the plasma membrane, as assayed by IGF-1 binding analysis and by Western blotting of plasma membrane proteins. The dolichyl phosphate-stimulated receptors were proven to be biochemically active since they exhibited autophosphorylation. Under normal conditions MDA231 cells, expressing very few IGF-1R at the cell surface, were not growth-arrested by an antibody (alphaIR-3) blocking the binding of IGF-1 to IGF-1R. However, after treatment with dolichyl phosphate, leading to a high cell surface expression of IGF-1R, alphaIR-3 efficiently blocked MDA231 cell growth. Taken together with the fact that the breast cancer cells produce IGF-1 and exhibit intracellular binding, our data suggest that the level of de novo -synthesized dolichyl phosphate may be critical for whether the cells will use an intracellular or an extracellular autocrine IGF-1 pathway.

Treatment with erythropoietin (epo) may improve the anemia of myelodysplastic syndromes (MDS) in approximately 20% of patients. Previous studies have suggested that treatment with the combination of granulocyte colony-stimulating factor (G-CSF) and epo may increase this response rate. In the present phase II study, patients with MDS and anemia were randomized to treatment with G-CSF + epo according to one of two alternatives; arm A starting with G-CSF for 4 weeks followed by the combination for 12 weeks, and arm B starting with epo for 8 weeks followed by the combination for 10 weeks. Fifty evaluable patients (10 refractory anemia [RA], 13 refractory anemia with ring sideroblasts [RARS], and 27 refractory anemia with excess blasts [RAEB]) were included in the study, three were evaluable only for epo as monotherapy and 47 for the combined treatment. The overall response rate to G-CSF + epo was 38%, which is identical to that in our previous study. The response rates for patients with RA, RARS, and RAEB were 20%, 46%, and 37%, respectively. Response rates were identical in the two treatment groups indicating that an initial treatment with G-CSF was not neccessary for a response to the combination. Nine patients in arm B showed a response to the combined treatment, but only three of these responded to epo alone. This suggests a synergistic effect in vivo by G-CSF + epo. A long-term follow-up was made on 71 evaluable patients from both the present and the preceding Scandinavian study on G-CSF + epo. Median survival was 26 months, and the overall risk of leukemic transformation during a median follow-up of 43 months was 28%. Twenty patients entered long-term maintenance treatment and showed a median duration of response of 24 months.The international prognostic scoring system (IPSS) was effective to predict survival, leukemic transformation, and to a lesser extent, duration of response, but had no impact on primary response rates.

OBJECTIVE: To evaluate the utilization of fine needle aspiration (FNA) biopsy to obtain material for reverse-transcriptase polymerase chain reaction (RT-PCR) in the detection of the t(X;18)(p11.2;q11.2) translocation in synovial sarcomas. STUDY DESIGN: We applied RT-PCR to detection of synovial sarcoma fusion gene transcripts on fine needle aspirates. Five clinical samples were first analyzed: one was a tumor previously diagnosed as malignant hemangiopericyloma, one was a poorly defined tumor, and three were suspected synovial sarcomas, FNA material was transferred directly to the RT-PCR reaction tube without RNA extraction. RESULTS: The t(X;18) translocation could be detected on the limited amount of material that FNA provides. In each of the cases studied the representivity of the tumor samples was confirmed microscopically. CONCLUSION: Our protocol permits analysis directly on representative samples without extraction of RNA. The results imply that RT-PCR offers reliable detection of sarcoma fusion gene transcripts on fine needle aspirates. The procedure, apart from being applicable to outpatients, is rapid and sensitive.

A study of bone marrow morphology and apoptosis was undertaken in 51 patients with myelodysplastic syndromes (MDS) treated with granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO). In 19 of these patients (37%), a significant improvement in the hemoglobin level was found after treatment. Apoptosis was measured using a nick-end labeling (TUNEL) technique. Patients with MDS had a significantly higher percentage of labelled (apoptotic) cells in the bone marrow compared to healthy individuals (56.3 +/- 3.8% vs. 16.2 +/- 1.4%, p = 0.0001). Patients with RAS showed a lower percentage of apoptotic cells than patients with RA (68.5 +/- 9% vs. 46.5 +/- 4.8%, p < 0.05), while patients with RAEB did not differ significantly from either RA or RAS. In the patients who responded to treatment, the bone marrow samples displayed significant morphological changes. The percentages of erythroid precursors and myeloblasts were reduced after treatment, and patients who had ring sideroblasts before treatment also showed a reduction in the percentage of these cells. Total erythroid index also decreased in responding patients. The percentage of apoptotic cells decreased significantly in responding patients (58.8 +/- 4.8% before treatment vs. 44.5 +/- 5.5% after treatment, mean reduction 18.3%, p = 0.0003), whereas no significant change was found in non-responding patients. Our results suggest that one important mechanism behind the positive effects of treatment with G-CSF and EPO is a reduction in the degree of ineffective hematopoiesis in MDS.

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.

About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen., Many patients with breast cancer develop resistance to the drug tamoxifen and relapse. Here Johansson et al. identify the nuclear receptor retinoic acid receptor alpha (RARA) as a marker of tamoxifen resistance and show that RARA expression correlates negatively with relapse-free survival of patients.

A patient having familiar adenomatosis polyposis and an ileo-rectal anastomosis developed a flat mucosal lesion in the rectum. A punch biopsy revealed a villous adenoma with high-grade dysplasia. The subsequent surgical specimen indicated that the flat villous adenoma was rich in Paneth cells. Special stains included lysozyme muramidase (to visualize Paneth cells), MIB1 proliferation monoclonal antibody and single and multilabel immunohistochemistry for Paneth cells. Other methods included transmission electron microscopy and quantification with an image quantifier (Program Optilab 2.1) of lysozyme-stained Paneth cells. The subjective evaluation of hematoxylin-eosin-stained preparations demonstrated that the Paneth cells were mainly located in the lower half of the villi. Sections labeled with a specific stain (lysozyme muramidase) revealed more Paneth cells in the villi and electron microscopy showed even more in lysozyme-negative areas. Obviously some migrating dysplastic Paneth cells had retained their characteristic granules on their way towards the tip of the villi. Quantitative studies indicated that the lysozyme muramidase-positive material accounted for 41% of the adenomatous tissue. MIB1 revealed intense cell proliferation at the base of the adenoma and in the entire slopes of the villi. Despite the wide distribution of Paneth cells in intestinal metaplasia of the stomach, in the normal small intestine and in the large bowel with chronic inflammatory diseases, it is surprising that tumors arising in Paneth cells are extremely rare. The causes of the apparent natural resistance of Paneth cells to tumor development deserve to be investigated. This is the first case of Paneth cell-rich flat adenoma of the rectum in the literature.

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.

The incidence of cervical adenocarcinoma, which accounts for 10-20% of all cervical cancers, has increased continuously in developed countries during the last two decades, unlike squamous cell cervical carcinoma. This increasing trend, noted particularly among women under the age of 40 years, has occurred despite extensive cytological Pap smear screening. A deeper understanding of the etiology of cervical adenocarcinoma, better preventive measures and reliable prognostic markers are urgently needed. The human leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family includes: LRIG1, LRIG2 and LRIG3. LRIG expression has proven to be of prognostic value in different types of human cancers, including breast cancer, early stage invasive squamous cervical cancer, cutaneous squamous cell carcinoma, oligodendroglioma and astrocytoma. LRIG1 functions as a tumor suppressor, while less is known about the functions of LRIG2 and LRIG3. This study evaluated the expression of the three LRIG proteins in tumor specimens from 86 women with pure cervical adenocarcinoma by immunohistochemistry. Possible correlations between LRIG expression and known prognostic factors, including human papillomavirus (HPV) status, FIGO stage and histology were investigated. Patient survival data were collected retrospectively and the possible prognostic value of LRIG protein expression was investigated. High staining intensity of LRIG1 and high fraction of LRIG3-positive cells were significantly associated with patient survival, and positive correlations were found between LRIG1 and LRIG3 staining intensity and HPV status. Thus, the LRIG proteins may be important determinants of cervical adenocarcinoma progression and their diagnostic and prognostic potential should be studied further.