High number of kinome-mutations in non-small cell lung cancer is associated with reduced immune response and poor relapse-free survival.

Lung cancer is the leading cause of cancer related death, and the past years' improved insight into underlying molecular events has significantly improved outcome for specific subsets of patients. In particular, several new therapies that target protein kinases have been implemented, and many more are becoming available. We have investigated lung cancer specimens for somatic mutations in a targeted panel of 612 human genes, the majority being protein kinases. The somatic mutation profiles were correlated to profiles of immune cell infiltration as well as relapse-free survival. Targeted deep sequencing was performed on 117 tumour/normal pairs using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes targeting 3.2 Mb of the human genome, including exons and untranslated regions of all known kinases, kinase receptors and selected cancer-related genes (612 genes in total). CD8 staining was determined using Ventana Benchmark. Survival analyses were performed using SPSS. The number of mutations per sample ranged from 0 to 50 (within the 612 genes tested), with a median of nine. The prognosis was worse for patients with more than the median number of mutations. A significant correlation was found between mutations in one of selected DNA-repair genes and the total number of mutations in that tumour (p < 0.001). There was a significant inverse correlation between the number of infiltrating stromal CD8+ lymphocytes and the presence of EGFR mutations.

Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumour, predominantly affecting children and adolescents. Cancer cell line models are required to understand the underlying mechanisms of tumour progression and for preclinical investigations. METHODS: To identify cell lines that are well suited for studies of critical cancer-related phenotypes, such as tumour initiation, growth and metastasis, we have evaluated 22 osteosarcoma cell lines for in vivo tumorigenicity, in vitro colony-forming ability, invasive/migratory potential and proliferation capacity. Importantly, we have also identified mRNA and microRNA (miRNA) gene expression patterns associated with these phenotypes by expression profiling. RESULTS: The cell lines exhibited a wide range of cancer-related phenotypes, from rather indolent to very aggressive. Several mRNAs were differentially expressed in highly aggressive osteosarcoma cell lines compared with non-aggressive cell lines, including RUNX2, several S100 genes, collagen genes and genes encoding proteins involved in growth factor binding, cell adhesion and extracellular matrix remodelling. Most notably, four genes-COL1A2, KYNU, ACTG2 and NPPB-were differentially expressed in high and non-aggressive cell lines for all the cancer-related phenotypes investigated, suggesting that they might have important roles in the process of osteosarcoma tumorigenesis. At the miRNA level, miR-199b-5p and mir-100-3p were downregulated in the highly aggressive cell lines, whereas miR-155-5p, miR-135b-5p and miR-146a-5p were upregulated. miR-135b-5p and miR-146a-5p were further predicted to be linked to the metastatic capacity of the disease. INTERPRETATION: The detailed characterisation of cell line phenotypes will support the selection of models to use for specific preclinical investigations. The differentially expressed mRNAs and miRNAs identified in this study may represent good candidates for future therapeutic targets. To our knowledge, this is the first time that expression profiles are associated with functional characteristics of osteosarcoma cell lines.

Patterns of genomic evolution in advanced melanoma.

Genomic alterations occurring during melanoma progression and the resulting genomic heterogeneity between metastatic deposits remain incompletely understood. Analyzing 86 metastatic melanoma deposits from 53 patients with whole-exome sequencing (WES), we show a low branch to trunk mutation ratio and little intermetastatic heterogeneity, with driver mutations almost completely shared between lesions. Branch mutations consistent with UV damage indicate that metastases may arise from different subclones in the primary tumor. Selective gain of mutated BRAF alleles occurs as an early event, contrasting whole-genome duplication (WGD) occurring as a late truncal event in about 40% of cases. One patient revealed elevated mutational diversity, probably related to previous chemotherapy and DNA repair defects. In another patient having received radiotherapy toward a lymph node metastasis, we detected a radiotherapy-related mutational signature in two subsequent distant relapses, consistent with secondary metastatic seeding. Our findings add to the understanding of genomic evolution in metastatic melanomas.

Analysis of the miR-34 family functions in breast cancer reveals annotation error of miR-34b.

The microRNAs in the miR-34 family, consisting of miR-34a, miR-34b and miR-34c, are tumour suppressors. The annotated human miR-34b-5p has one additional base at the 5' end of the common miR-34 family seed sequence, compared to miR-34a-5p and miR-34c-5p. This extra base results in a shift of the seed sequence, which would affect the target gene repertoire and have functional consequences. During our studies of miR-34 functions, we investigated the precise sequence of mature miR-34b-5p in human cells by deep sequencing. We found that a miR-34b-5p without the extra base was the predominant form in both non-malignant and malignant cells derived from several human tissues, indicating that the miR-34b annotation is misleading. We evaluated the functional implications of the seed shift, by comparing the effect of mimics representing the alternative miR-34b-5p sequences in MDA-MB-231 cells. In contrast to the annotated miR-34b, the endogenously expressed miR-34b displayed tumour suppressive characteristics in vitro similarly to miR-34c. These data demonstrate the importance of determining the precise sequence of a mature microRNA before exploring miRNA functions.

Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas.

Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkin's lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2-18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.

MDM2 gene amplification and transcript levels in human sarcomas: relationship to TP53 gene status.

BACKGROUND: Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of human cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein that may interact with both mutant and wild-type p53 proteins, thereby inhibiting p53-mediated transactivation in a dose-dependent manner. Recently it has been suggested that mdm2 and p53 proteins are components of an autoregulatory loop in which the MDM2 gene is transactivated by p53. PURPOSE: Our purpose was to examine the frequency of MDM2 amplifications in larger panels of sarcomas, determine if the mRNA level could be elevated in tumors without concomitant gene amplification, and relate MDM2 findings to the TP53 status of the tumors. METHODS: Sarcoma tissue of different histologic subtypes was obtained from 68 patients at the time of surgery and from 26 human xenografts in nude mice. In addition, two human sarcoma cell lines (OSA and U2OS) were studied. Genomic DNA from tumor tissue, in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mutations in exons 5, 7, and 8 by constant denaturing gel electrophoresis. To determine the MDM2 and TP53 mRNA levels, Northern-blot analysis was performed. RESULTS: Amplification of the MDM2 gene was detected in 10 tumors (10.3%). Whereas MDM2 amplification and/or over-expression were found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, one of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 leiomyosarcomas, such alterations were observed in two of two fibrosarcomas, three of six malignant schwannomas, three of 19 liposarcomas, and in the one hemangiopericytoma examined. MDM2 overexpression was found in all nine examined cases with and in three tumors without amplification. TP53 mutations were detected in 12 cases (five osteosarcomas, four MFHs, and three leiomyosarcomas), of which none showed amplification, but one had increased levels of MDM2 mRNA. None of the fibrosarcomas, malignant schwannomas, and liposarcomas examined had mutated TP53. The six sarcomas that showed high TP53 mRNA expression in the absence of gene mutation also had elevated levels of MDM2 mRNA. CONCLUSIONS: The present data provide further indications that increased MDM2 expression level, caused by gene amplification or altered regulation of transcription, is involved in tumor progression of some, but not all, sarcoma subtypes.

Expression of the neuroectodermal intermediate filament nestin in human melanomas.

Nestin is a newly identified intermediate filament expressed in proliferating neuronal progenitor cells, but not in the adult brain. Nestin expression reappears in many tumors of the central nervous system and has in human glioblastomas been associated with a high degree of malignancy. Because melanocytes are of neuroectodermal origin, we studied nestin expression in benign and malignant cells of the melanocytic lineage using Northern blot and immunohistochemical analyses. Nestin mRNA was detected in 24 of 34 metastatic melanomas and in 1 of 4 benign nevi, whereas the protein was expressed in 10 of 15 primary melanomas, in 29 of 34 metastatic tumors, and in 3 of 4 nevi. Neither normal melanocytes nor any of 4 basal cell carcinomas showed detectable levels of the protein. The high fraction of melanocytic tumors which express nestin, particularly the metastatic melanomas, suggests that nestin may be a useful marker for such malignancies. Furthermore, although no significant correlation between nestin expression and tumor malignancy was observed, the protein was most abundantly expressed in the infiltrating part of the tumors, indicating a possible involvement of nestin in tumor invasion.

Levels of nm23 messenger RNA in metastatic malignant melanomas: inverse correlation to disease progression.

Data obtained in experimental murine tumors and in clinical specimens of human breast cancer have suggested that the nm23 gene may function as a metastasis suppressor gene. In this report we examined the nm23 mRNA level in tumor tissue obtained from distant metastases in 33 patients with malignant melanoma. The gene was differentially expressed in the tumors with a 20-fold range in hybridization intensities. The levels of nm23 mRNA in benign nevi obtained from 12 of the 33 patients were relatively low, with a mean value of 17% of that in the melanomas. In attempts to relate the level of nm23 expression in the tumor metastases to progression of the disease, the time from biopsy of the primary tumor to the appearance of metastases was used as a clinical end point. It was found that patients developing metastases during the first 2 years after diagnosis had significantly lower levels of tumor nm23 expression (56% of the mean value) compared to patients with less aggressive disease (164%) (P < 0.0004). In concordance with previous data the association found here between low levels of nm23 mRNA and the malignant potential of melanomas suggests that the nm23 gene may be implicated in the mechanism of disease progression in some types of human cancer.

p53 abnormalities in different subtypes of human sarcomas.

In this report we examined p53 alterations at the DNA, mRNA, and protein levels on tissue from 39 patients with different subtypes of sarcoma. Loss of heterozygosity for the chromosome 17p region was found in 60, 63, and 33% of 10 informative osteosarcomas, 11 malignant fibrous histiocytomas, and 6 leiomyosarcomas, respectively. In addition, 2 of 10 tumors belonging to a heterogeneous group of soft tissue sarcomas showed loss of heterozygosity. Elevated levels of p53 mRNA were found in six tumors, four had a truncated transcript, and in six patients no mRNA was detected. In most cases, elevated transcript levels were accompanied by overexpression of protein as studied by immunohistochemistry, whereas the presence of truncated transcripts was associated with negative immunostaining. Point mutations in exons 5, 7, or 8 of the TP53 gene were detected in seven tumors. Six of these expressed high levels of mRNA and protein, probably reflecting a point mutation in one of the alleles and loss of the other. Three of the mutations have not previously been described. Taken together, p53 abnormalities were found in approximately 65% of the osteosarcomas, malignant fibrous histiocytomas, and leiomyosarcomas examined and in 30% of the other soft tissue tumors. The results indicate that the TP53 gene is involved in the tumorigenesis of several sarcoma subtypes in a higher fraction of cases than was previously recognized.

Reversal of the in vivo metastatic phenotype of human tumor cells by an anti-CAPL (mts1) ribozyme.

The putative role of the CAPL gene in enhancing the development of human cancer metastasis was examined by transfecting human high-expressing osteosarcoma cells with a hammerhead ribozyme directed against the gene transcript. The ability of the ribozyme to cleave target mRNA in intact cells was demonstrated in a 5'-rapid amplification of cDNA ends assay. In transfected cells, a suppression of the capacity to give skeletal metastases upon intracardial injection into nude rats was observed in cell clones with reduced expression of CAPL mRNA and protein, whereas in vitro and in vivo cell proliferation and tumorigenicity were unchanged. The results provide direct evidence that the expression level of the CAPL-encoded protein can determine the metastatic potential of osteosarcoma cells, and they demonstrate an association between reduced gene expression and proliferation-independent inhibition of the metastatic capacity of human tumor cells. The effects of the specific cleavage of CAPL mRNA indicate that the gene product is involved in key cellular functions associated with the metastatic process and suggest that therapeutic modulation of the protein function may represent a novel approach for inhibiting the metastatic spread of cancer cells.

HMGIC, the gene for an architectural transcription factor, is amplified and rearranged in a subset of human sarcomas.

Amplified segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate amplified regions around the MDM2 and CDK4 genes. Here we show recurrent amplification of a third region encompassing HMGIC, a human architectural transcription factor gene. Reduced amplification frequency of sequences flanking the gene was observed, indicating that inclusion of this third region in the amplicons is also selected for. In three samples only the 5' part of HMGIC was amplified, suggesting preferential loss of the 3' part of the gene preceding or during amplification. In several other samples rearrangement of the gene was observed. Expression analysis showed transcripts of aberrant sizes, lacking 3' sequences, and 3' RACE of one sample revealed replacement of exons 4 and 5 with ectopic sequences. This truncation of HMGIC resembles that reported for translocations of HMGIC in benign tumors, including lipomas, and it is striking that the gene was frequently amplified or rearranged in well differentiated liposarcomas, the malignant counterpart of lipomas. It seems conceivable that high levels of either full length or truncated hmgic could be relevant for the etiology of these tumors.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

BRAF V600E mutation in early-stage multiple myeloma: good response to broad acting drugs and no relation to prognosis.

In this study, we analyzed the prevalence and clone size of BRAF V600E mutation in 209 patients with multiple myeloma and related the results to clinical phenotype, response and survival. Biopsies were screened for BRAF V600E by allele-specific real-time PCR (AS-PCR). Positive results were confirmed by immunohistochemistry, Sanger sequencing and, in three patients from whom we had stored purified myeloma cells, whole-exome sequencing. Eleven patients (5.3%) were BRAF V600E mutation positive by AS-PCR and at least one other method. The fraction of mutated cells varied from 4 to 100%. BRAF V600E-positive patients had no characteristic clinical phenotype except for significantly higher levels of serum creatinine (125 versus 86 µmol/l) Seven of eleven patients responded with at least very good partial response to alkylators, immunomodulatory agents or proteasome inhibitors. Progression-free and overall survival were similar in patients with and without the mutation. By this integrated approach, we found that patients with BRAF V600E mutation responded very well to broad acting drugs and there was no relation to prognosis in early-stage myeloma. In particular, a large mutated cell fraction did not correlate with aggressive disease.

Cloning and characterization of two forms of bovine polymeric immunoglobulin receptor cDNA.

The polymeric immunoglobulin receptor (transmembrane secretory component) mediates transcellular transport of dimeric immunoglobulin A (IgA) and pentameric IgM in glandular and mucosal epithelial cells. cDNAs encoding two forms of the bovine polymeric immunoglobulin receptor (pIgR) have been cloned and sequenced. The long form contains 3,527 bp and predicts a single open reading frame of 2,271 bp encoding a protein of 757 bp. The extracellular part contains five immunoglobulin (Ig)-like domains. The shorter form lacks the region from residues 458-1,111 corresponding to Ig-like domains 2 and 3. In Northern blot analysis of various bovine tissues, only the long form of pIgR mRNA was detected. By using the reverse transcription-polymerase chain reaction (RT-PCR), both forms were detected. An alignment of the cytoplasmic tail of the pIgR from bovine, human, rabbit, and rat revealed highly conserved areas that may reflect the importance of these regions for intracellular sorting of the receptor.

The protooncogene CHOP/GADD153, involved in growth arrest and DNA damage response, is amplified in a subset of human sarcomas.

The C/EBP-homologous transcription factor CHOP (GADD153) is inducible by growth inhibition or DNA damage, and has been shown to be oncogenically activated by the specific (12;16) translocation in human myxoid liposarcoma. We have now found CHOP amplification in two sarcoma cell lines with previously reported amplification of the nearby GLI gene. Among 98 other human sarcomas of various types, CHOP was amplified in a hemangiopericytoma, a liposarcoma, and two osteosarcoma. High constitutive expression levels of CHOP were observed in tumors with gene amplification, but also in some other samples. The nearby MDM2 gene, which codes for a protein that may inactivate wild-type p53, has previously been reported to be frequently amplified in sarcoma. In our sarcoma panel, MDM2 was amplified in 9 cases. MDM2 and CHOP were co-amplified in two of these, whereas the two osteosarcomas had amplified CHOP but not MDM2. CHOP was amplified in both cell lines with GLI amplification, and MDM2 only in one. No mutations in the TP53 gene have been found in samples with amplification of MDM2. In contrast, the cell line in which CHOP but not MDM2 was amplified had mutated TP53, suggesting that selection of this amplicon was not mediated through p53 inactivation.

Identification of two distinct chromosome 12-derived amplification units in neuroblastoma cell line NGP.

The neuroblastoma cell line NGP contains two homogeneously staining regions (hsr). One of these hsrs contains MYCN sequences. Reverse painting experiments demonstrated that the second HSR consisted of two chromosome 12-derived amplification units, located at 12q14-15 and 12q24. Southern blot and fluorescence in situ hybridization (FISH) analysis showed amplification of genes located at 12q14-15: SAS, MDM2, and CDK4, GLI, CHOP, CDK2, and A2MR were found not to be amplified. FISH further demonstrated amplification of RSN, a gene located at 12q24. The finding of two distinct chromosome 12 amplification units in a neuroblastoma cell line NGP is reminiscent of recent findings in well-differentiated liposarcoma (WDLPS) and other sarcomas. The second amplification unit on chromosome 12 in NGP is located more distal (12q24) than the one observed in WDLPS (12q21). The mechanism and biologic significance of this amplification process in neuroblastoma and WDLPS remain to be elucidated.

A well-differentiated liposarcoma with a new type of chromosome 12-derived markers.

Well-differentiated liposarcomas (WDLPS) are cytogenetically characterized by the presence of supernumerary ring or giant rod marker chromosomes. These supernumerary chromosomes are composed of amplified sequences from chromosome 12 (12q14 approximately 15) in association with amplified segments from various other chromosomes, and contain alterations of the alpha satellite sequences. We report a case of WDLPS of the lipoma-like and sclerosing subtype that contains a novel type of supernumerary marker chromosome. Instead of rings or giant rods, these cells had three apparently identical copies of a subtelocentric supernumerary marker with a size and shape similar to C-group chromosomes. Fluorescence in situ hybridization analysis revealed that the markers were composed of amplified material from 12q14 approximately 15, including the genes MDM2 and CDK4. Similar to the rings and giant rods observed in other WDLPS cases, these unusual markers had no alpha satellite repeats at the primary constriction site, but centromeric activity could be demonstrated by using anti-centromere protein C antibodies. These findings show that the supernumerary markers of WDLPS may be variable in size and shape, but consistently share the same genomic structure, specifically 12q amplified sequences together with centromere alterations, and underline the importance of molecular methods in the diagnosis of adipose tissue tumors.

Dedifferentiation of a well-differentiated liposarcoma to a highly malignant metastatic osteosarcoma: amplification of 12q14 at all stages and gain of 1q22-q24 associated with metastases.

Well-differentiated liposarcomas (WDLPS), especially those located in the retroperitoneum, may occasionally undergo dedifferentiation. Although this process is associated with a more aggressive clinical course, dedifferentiated liposarcomas rarely produces metastases. The case reported here is rather uncommon: A retroperitoneal WDLPS gave lung metastases that were diagnosed as highly malignant osteosarcomas. We used comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and Southern blot analyses to characterize the copy number changes and genetic aberrations occurring at different stages of the disease. In the primary tumor, the only detectable aberration was amplification of 12q13-q14, which was present only in a fraction of the cells and revealed by FISH analysis. High-level amplification of 12q13-q14, involving CDK4, MDM2, and HMGIC, was seen both in the relapse and the metastases. The second most common change, gain or high-level amplification of 1q22-q24, was detectable by CGH only in the osteogenic metastases, as was loss of the distal 2q. FISH analyses revealed considerable heterogeneity in the samples, and the percentage of cells showing aberrations was significantly higher in the metastatic samples. In particular, increased copy numbers of 789f2, a marker for 1q21 amplification in sarcomas, was observed in more than 65% of the cells in the metastatic samples, but in less than 10% of the cells from the recurrent samples. These observations could indicate that 1q amplification, in particular, may be indicative of a more malignant phenotype and ability of metastasis in WDLPS, as has also been suggested by others.

Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma.

To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23-qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53-associated cell cycle regulatory pathways may play prominent roles in the development of human MFH.