A stress-induced early innate response causes multidrug tolerance in melanoma.

Correction to: Oncogene (2015) 34, 4448­4459; doi:10.1038/onc.2014.372; published online 24 November 2014. In this article, published online 24 November 2014, the authors have noticed that the latest supplementary information was not used. The corrected supplementary information (Supplementary Materials) appears online together with this corrigendum. The authors would like to apologise for any inconvenience this may cause

A stress-induced early innate response causes multidrug tolerance in melanoma.

Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

MiR-200a regulates epithelial to mesenchymal transition-related gene expression and determines prognosis in colorectal cancer patients.

BACKGROUND: MicroRNAs (miRNAs) regulate the biological properties of colorectal cancer (CRC) cells and might serve as potential prognostic factors and therapeutic targets. In this study, we therefore globally profiled miRNAs associated with E-cadherin expression in CRC cells in an attempt to identify miRNAs that are associated with aggressive clinical course in CRC patients. METHODS: Two CRC cell lines (Caco-2 and HRT-18) with different E-cadherin expression pattern were profiled for differences in abundance for more than 1000 human miRNAs using microarray technology. One of the most differentially expressed miRNAs, miR-200a was evaluated for its prognostic role in a cohort of 111 patients and independently validated in 217 patients of the Cancer Genome Atlas data set. To further characterise the biological role of miR-200a expression in CRC, in vitro miR-200a inhibition and overexpression were performed and the effects on cellular growth, apoptosis and epithelial-mesenchymal transition (EMT)-related gene expression were explored. RESULTS: In situ hybridisation specifically localised miR-200a in CRC cells. In both cohorts, a low miR-200a expression was associated with poor survival (P<0.05). Multivariate Cox regression analysis identified low levels of miR-200a expression as an independent prognostic factor with respect to cancer-specific survival (HR=2.04, CI=1.28-3.25, P<0.002). Gain and loss of function assays for miR-200a in vitro led to a significantly differential and converse expression of EMT-related genes (P<0.001.) A low expression of miR-200a was also observed in cancer stem cell-enriched spheroid growth conditions (P<0.05). CONCLUSIONS: In conclusion, our data suggest that low miR-200a expression is associated with poor prognosis in CRC patients. MiR-200a has a regulatory effect on EMT and is associated with cancer stem cell properties in CRC.

Validation of C-reactive protein levels as a prognostic indicator for survival in a large cohort of pancreatic cancer patients.

BACKGROUND: Recent evidence indicates that the host inflammatory response has an important role in the tumour progression. Elevated C-reactive protein (CRP) levels have been previously associated with poor prognosis in several cancer types including small-scale studies in pancreatic cancer (PC) patients. The purpose of the present study was to validate the prognostic impact of plasma CRP levels at date of diagnosis on cancer-specific survival (CSS) in a large cohort of PC patients. METHODS: Data from 474 consecutive patients with adenocarcinoma of the pancreas, treated between 2004 and 2012 at a single centre, were evaluated retrospectively. CSS was analysed using the Kaplan-Meier method. To evaluate the prognostic significance of plasma CRP levels, univariate and multivariate Cox analyses were applied. RESULTS: High plasma CRP levels at diagnosis were significantly associated with well-established prognostic factors, including high tumour stage and tumour grade and the administration of chemotherapy (P<0.05). In univariate analysis, we observed that a high plasma CRP level was a consistent factor for poor CSS in PC patients (hazard ratio (HR)=2.21; 95% confidence interval (CI)=1.68-2.92, P<0.001). In multivariate analysis, tumour stage, grade, administration of chemotherapy, a high neutrophil-lymphocyte ratio and the highest quartile of CRP levels (HR=1.60, 95% CI=1.16-2.21; P=0.005) were identified as independent prognostic factors in PC patients. CONCLUSION: In conclusion, we confirmed a significant association of elevated CRP levels with poor clinical outcome in PC patients. Our results indicate that the plasma CRP level might represent a useful marker for patient stratification in PC management.

Report from the OECI Oncology Days 2014.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe.

Increased neutrophil-lymphocyte ratio is a poor prognostic factor in patients with primary operable and inoperable pancreatic cancer.

BACKGROUND: The neutrophil-lymphocyte ratio (NLR) has been proposed as an indicator of systemic inflammatory response. Previous findings from small-scale studies revealed conflicting results about its independent prognostic significance with regard to different clinical end points in pancreatic cancer (PC) patients. Therefore, the aim of our study was the external validation of the prognostic significance of NLR in a large cohort of PC patients. METHODS: Data from 371 consecutive PC patients, treated between 2004 and 2010 at a single centre, were evaluated retrospectively. The whole cohort was stratified into two groups according to the treatment modality. Group 1 comprised 261 patients with inoperable PC at diagnosis and group 2 comprised 110 patients with surgically resected PC. Cancer-specific survival (CSS) was assessed using the Kaplan-Meier method. To evaluate the independent prognostic significance of the NLR, the modified Glasgow prognostic score (mGPS) and the platelet-lymphocyte ratio univariate and multivariate Cox regression models were applied. RESULTS: Multivariate analysis identified increased NLR as an independent prognostic factor for inoperable PC patients (hazard ratio (HR)=2.53, confidence interval (CI)=1.64-3.91, P<0.001) and surgically resected PC patients (HR=1.61, CI=1.02-2.53, P=0.039). In inoperable PC patients, the mGPS was associated with poor CSS only in univariate analysis (HR=1.44, CI=1.04-1.98). CONCLUSION: Risk prediction for cancer-related end points using NLR does add independent prognostic information to other well-established prognostic factors in patients with PC, regardless of the undergoing therapeutic modality. Thus, the NLR should be considered for future individual risk assessment in patients with PC.

KRAS mutation analysis on low percentage of colon cancer cells: the importance of quality assurance.

KRAS mutation testing is mandatory for patients with metastatic colorectal cancer who are eligible for treatment with an epidermal growth factor receptor targeting agent, since tumors with a mutation are not sensitive to the drug. Several methods for mutation testing are in use and the need for external quality assurance has been demonstrated. An often little addressed but important issue in external quality assurance schemes is a low percentage of tumor cells in the test samples, where the analytical sensitivity of most tests becomes critical. Using artificial samples based on a mixture of cell lines with known mutation status of the KRAS gene, we assessed the reliability of a series of commonly used methods (Sanger sequencing, high resolution melting, pyrosequencing, and amplification refractory mutation system-polymerase chain reaction) on samples with 0, 2.5, 5, 10, and 15 % mutated cells. Nine laboratories throughout Europe participated and submitted a total of ten data sets. The limit of detection of each method differed, ranging from >15-5 % tumor cells. All methods showed a decreasing correct mutation call rate proportionally with decreasing percentage of tumor cells. Our findings indicate that laboratories and clinicians need to be aware of the decrease in correct mutation call rate proportionally with decreasing percentage of tumor cells and that external quality assurance schemes need to address the issue of low tumor cell percentage in the test samples.

Overexpression of the anti-apoptotic protein AVEN contributes to increased malignancy in hematopoietic neoplasms.

AVEN has been identified as an inhibitor of apoptosis, which binds to the adaptor protein, APAF-1, and thereby prevents apoptosome formation and mitochondrial apoptosis. Recent data have demonstrated high expression levels of AVEN messenger RNA in acute leukemias as well as a positive correlation between AVEN mRNA overexpression and poor prognosis in childhood acute lymphoblastic leukemia. On the basis of these data, we investigated the potential involvement of AVEN in tumorigenesis. First, we confirmed the overexpression of AVEN in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patient samples. We then established a transgenic mouse model with T-cell-specific overexpression of AVEN, with which we demonstrated the oncogenic cooperation of AVEN with heterozygous loss of p53. Finally, we used a subcutaneous xenograft mouse model to show that AVEN knockdown in the T-ALL cell lines, MOLT-4 and CCRF-CEM, and in the acute myeloblastic leukemia cell line, Kasumi-1, leads to a halt in tumor growth owing to the increased apoptosis and decreased proliferation of tumor cells. Collectively, our data demonstrate that the anti-apoptotic molecule, AVEN, functions as an oncoprotein in hematopoietic neoplasms.

Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity.

According to the concept of lipotoxicity, ectopic accumulation of lipids in non-adipose tissue induces pathological changes. The most prominent effects are seen in fatty liver disease, lipid cardiomyopathy, non-insulin-dependent diabetes mellitus, insulin resistance and skeletal muscle myopathy. We used the MCK(m)-hLPL mouse distinguished by skeletal and cardiac muscle-specific human lipoprotein lipase (hLPL) overexpression to investigate effects of lipid overload in skeletal muscle. We were intrigued to find that ectopic lipid accumulation induced proteasomal activity, apoptosis and skeletal muscle damage. In line with these findings we observed reduced Musculus gastrocnemius and Musculus quadriceps mass in transgenic animals, accompanied by severely impaired physical endurance. We suggest that muscle loss was aggravated by impaired muscle regeneration as evidenced by reduced cross-sectional area of regenerating myofibers after cardiotoxin-induced injury in MCK(m)-hLPL mice. Similarly, an almost complete loss of myogenic potential was observed in C2C12 murine myoblasts upon overexpression of LPL. Our findings directly link lipid overload to muscle damage, impaired regeneration and loss of performance. These findings support the concept of lipotoxicity and are a further step to explain pathological effects seen in muscle of obese patients, patients with the metabolic syndrome and patients with cancer-associated cachexia.

Down-regulation of KRAS-interacting miRNA-143 predicts poor prognosis but not response to EGFR-targeted agents in colorectal cancer.

BACKGROUND: MicroRNA-143 (miRNA-143) is frequently down-regulated in colorectal cancer (CRC) and may influence CRC cell proliferation, apoptosis and sensitivity to 5-fluorouracil. mRNA encoded by the KRAS oncogene has been identified as a target of miRNA-143. However, the prognostic significance of miRNA-143 expression and the ability to predict patient response to epidermal growth factor receptor (EGFR)-targeted agents have not yet been explored. METHODS: We examined 77 CRC patients who were identified by pyrosequencing to have wild-type KRAS and were subsequently treated with EGFR-targeted therapy with the monoclonal antibodies cetuximab or panitumumab. MicroRNA-143 expression was measured in CRC tissue and corresponding non-neoplastic colon tissue by RT-PCR and its expression level was correlated with clinico-pathological characteristics. Univariate and multivariate analyses were used to calculate cancer-specific survival (CSS). The progression-free survival (PFS) and objective response rates on EGFR-targeted therapy were also evaluated. RESULTS: Down-regulation of miRNA-143 was observed in 47 out of 77 (61%) tumours. Multivariate Cox regression analysis identified low levels of miRNA-143 expression as an independent prognostic factor with respect to CSS (hazard ratio=1.92, confidence interval=1.1-3.4, P=0.024). A significant difference was also observed with regard to PFS on EGFR-targeted therapy (P=0.031), but there were no significant differences with regard to the objective response rates. CONCLUSION: Our data indicate that miRNA-143 expression levels serve as an independent prognostic biomarker for CRC in KRAS wild-type patients. No role for miRNA-143 expression as a predictive biomarker for EGFR-targeted agents could be identified. Given its negative impact on CSS and PFS, miRNA-143 represents a novel prognosticator and a promising drug target for patients with CRC.

Loss of RAF kinase inhibitor protein is a somatic event in the pathogenesis of therapy-related acute myeloid leukemias with C-RAF germline mutations.

We recently described oncogenic and anti-apoptotic C-RAF germline mutations in patients with therapy-related acute myeloid leukemia (t-AML). Activation of the RAF effector ERK was restricted to transformed cells, suggesting the requirement for cooperating events in leukemogenesis. Western blot analysis of blast cells from patients with C-RAF germline mutations revealed loss of the tumor and metastasis suppressor RAF kinase inhibitor protein (RKIP). Immunohistochemistry of the patients' primary tumors revealed normal RKIP expression levels, indicating that the loss of RKIP is a somatic, t-AML-specific event. In focus formation assays, the oncogenic potential of human mutant C-RAF was strongly influenced by expression levels of RKIP. Although the number of colonies formed by C-RAF(S427G) was significantly increased by RKIP silencing, the opposite was observed after RKIP overexpression. These results show that the loss of RKIP is a functional somatic event in carriers of C-RAF germline mutations, which contributes to the development of t-AML.

KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program.

Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30-40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.

mRNA expression patterns indicate CD30 mediated activation of different apoptosis pathways in anaplastic large cell lymphoma but not in Hodgkin's lymphoma.

One of the main functions of the tumor necrosis factor receptor (TNFR) family is induction of apoptosis. CD30, a member of the TNFR superfamily is overexpressed in highly proliferating tumors such as anaplastic large cell lymphoma (ALCL) and Hodgkin's lymphoma (HL). CD30 stimulation leads to apoptosis and growth arrest in cultured ALCL, but not in Hodgkin-Reed-Sternberg cells. To identify changes in the transcriptional program responsible for these opposing effects, we performed gene expression analysis in CD30-stimulated ALCL (Karpas 299) and HL (KM-H2) cell lines using cDNA microarrays. Selected genes were validated by real-time PCR. Hierarchical clustering was applied to the whole dataset and separated the cell lines clearly with respect to their origin. In HL, there were only minor CD30-specific alterations, whereas ALCL unequivocally showed a pronounced CD30-specific transcriptional response. Ninety-three genes (6.6% of total) were deregulated by more than a factor of two after CD30 stimulation in ALCL cells. The majority of genes identified are involved in cell cycle regulation and apoptosis. mRNA expression patterns further indicate that in contrast to HL, CD30 stimulation in ALCL induces cell death via the CD95-CD95 ligand (CD95L) pathway and the TNF-R1/TNF-R2 crosstalk. These data provide a detailed view on the transcriptional changes upon CD30 stimulation and may explain the observed functional differences of HL and ALCL.

Critical issues in the identification and management of patients with hereditary non-polyposis colorectal cancer.

Inherited defects of the DNA mismatch repair system are the underlying cause of the hereditary non-polyposis colorectal cancer (HNPCC) syndrome and are responsible for 3-4% of all cases of colorectal cancer. The HNPCC syndrome also carries the risk of development of additional malignancies such as endometrial, stomach, small bowel, ovarian, pancreas, ureter, renal pelvis, biliary tract and brain tumours. Amsterdam I and II criteria have been developed to clinically identify affected families. The revised Bethesda criteria function to select patients whose tumours should be investigated for microsatellite instability, the molecular hallmark of defects of the DNA mismatch repair proteins such as hMLH1 and hMSH2. Microsatellite instability-positive cases should be investigated for germline defects in the respective genes. This facilitates identification of affected family members that have to be included in special surveillance programmes, while unaffected family members are spared the physical discomfort and psychological burden of cancer surveillance. In this article, strategies for effective clinical as well as genetic detection of affected individuals, surveillance and appropriate preventive measures are discussed. Open questions include the role of chemoprevention, preventive surgical procedures, new endoscopic procedures as well as non-invasive 'virtual colonoscopy' and the exact implications of some mutations of the DNA mismatch repair genes. Perhaps most importantly, efforts should be made to more efficiently transfer information about the HNPCC syndrome and the cancer risk associated with it from the specialists to primary health care providers and the general public.

Prognostic relevance of tumour-associated macrophages and von Willebrand factor-positive microvessels in colorectal cancer.

Tumour-associated macrophages (TAM) are involved in tumour angiogenesis and anti-tumour immune response. In colorectal cancer (CRC), an association of high microvascular density (MVD) and unfavourable prognosis has been reported by some investigators. However, heterogeneous patient groups were studied. We, therefore, analysed the correlation between TAM and MVD and the prognostic relevance of MVD, TAM and T lymphocyte infiltration for long-term survival in a homogeneous group of 70 patients with moderately differentiated cancers of the International Union Against Cancer (UICC) stages II and III, who did not receive chemotherapy. MVD was evaluated using immunohistochemistry with antibodies against CD34 and von Willebrand factor (vWF). TAM and T lymphocytes were visualised with antibodies against CD68 and CD3, respectively. Statistical analysis did not reveal a significant correlation between TAM and T lymphocyte numbers and MVD. Multivariate analysis of immunohistochemical data from all CRC patients and the subgroup of patients with UICC stage-II CRC identified TAM- and vWF-positive microvessel numbers as prognostically relevant markers. Low numbers of TAM- and high numbers of vWF-positive microvessels were associated with an unfavourable prognosis. In conclusion, TAM- and vWF-positive microvessel numbers may serve as independent prognostic markers for patients with UICC stage-II and -III CRC and may help to identify patients with an unfavourable prognosis.

Mutation analyses in 17 patients with deficiency in acid beta-galactosidase: three novel point mutations and high correlation of mutation W273L with Morquio disease type B.

An inherited deficiency in beta-galactosidase can result in GM1 gangliosidosis, with several phenotypes of generalized or chronic psychomotor deterioration, as well as in Morquio disease type B, a characteristic mucopolysaccharidosis free of neurological symptoms. We performed mutation analyses in 17 juvenile and adult patients from various European regions with a deficiency in beta-galactosidase and skeletal abnormalities. Fifteen of these had the Morquio B phenotype and have remained neurologically healthy until now while the two others exhibited psychomotor retardation of juvenile onset. A two-base substitution (851-852TG-->CT; W273L) was present in 14 of the 15 Morquio B cases. Even if one excludes alleles from patients with possible common descent, there was a much higher frequency (79%) among those with Morquio B phenotype for the W273L mutation than previously reported in the literature (37%). That the Morquio phenotype is also expressed in heterozygotes for W273L and alleles typically found in GM1 gangliosidosis makes it possible to predict the phenotype and reliably detect heterozygotes. A single French patient had a novel missense point mutation (Q408P) together with a known mutation (T500A) while the mentally retarded patients were both heterozygous for two mutations known in chronic GM1 gangliosidosis together with two novel missense point mutations (Y270D and H281Y) in the vicinity of W273L. Our results confirm the high impact of Trp 273 for the function of beta-galactosidase and the expression of the Morquio B phenotype. In addition, a second domain around the amino acids 400-500 may also be of significance.

Extramedullary T cell lymphoblastic transformation of chronic myeloid leukaemia successfully treated with matched unrelated donor bone marrow transplantation.

Chronic myeloid leukaemia (CML) inevitably terminates in blast crisis (BC) which is of myeloid phenotype in approximately two-thirds and B-lymphoid in one-third of patients. T cell BC is rare and associated with poor prognosis. We describe the case of a 48-year-old woman with extramedullary T cell lymphoblastic transformation. After treatment with combination chemotherapy she achieved a second chronic phase and underwent an allogeneic BMT from an HLA-matched unrelated donor. At 30 months follow-up she is still in complete molecular remission and in good clinical condition. We conclude that unrelated donor BMT should be considered as a therapeutic option for patients with extramedullary BC.

Nasopharyngeal angiofibroma: an APC-gene-associated tumor?

Nasopharyngeal angiofibromas are extremely rare, locally invasive tumors of unknown cause exclusively occurring in male adolescents. Recently, 6 cases have been reported in patients with familial adenomatous polyposis coli (Gardners syndrome). Mutations or allelic loss of the adenomatous polyposis coli (APC) gene have therefore been implied in the pathogenesis of nasopharyngeal angiofibroma. The authors analyzed 11 cases of nasopharyngeal angiofibromas from 9 male patients for mutations in the mutation cluster region and allelic loss of the APC gene. Six were primary tumors; 2 first recurrences; and 1, primary tumor with 2 recurrences. Direct sequence analysis was performed on several overlapping polymerase chain reaction (PCR) products. Detection of allelic loss was performed by restriction length polymorphism analysis at a polymorphic locus. No mutation was detected in 11 tumors of 9 different patients. None of the 9 informative (heterozygous) cases carried an allelic loss. We conclude that alterations of the APC gene do not play a major role in the development of nasopharyngeal angiofibroma. The coincidence of nasopharyngeal angiofibromas and adenomatous polyposis coli in some families implies the possibility that another gene in this region might be responsible for the development of nasopharyngeal angiofibromas. HUM PATHOL 31:1411:1413.