Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

PURPOSE: Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients. EXPERIMENTAL DESIGN: We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA). RESULTS: Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% (n = 163) and sensitivities of 78% (n = 23) and 67% (n = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, P = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade. CONCLUSIONS: These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade.See related commentary by Wang and Ajani, p. 6887.

Direct detection of early-stage cancers using circulating tumor DNA.

Early detection and intervention are likely to be the most effective means for reducing morbidity and mortality of human cancer. However, development of methods for noninvasive detection of early-stage tumors has remained a challenge. We have developed an approach called targeted error correction sequencing (TEC-Seq) that allows ultrasensitive direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing. We have used this approach to examine 58 cancer-related genes encompassing 81 kb. Analysis of plasma from 44 healthy individuals identified genomic changes related to clonal hematopoiesis in 16% of asymptomatic individuals but no alterations in driver genes related to solid cancers. Evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of 71, 59, 59, and 68%, respectively, of patients with stage I or II disease. Analyses of mutations in the circulation revealed high concordance with alterations in the tumors of these patients. In patients with resectable colorectal cancers, higher amounts of preoperative circulating tumor DNA were associated with disease recurrence and decreased overall survival. These analyses provide a broadly applicable approach for noninvasive detection of early-stage tumors that may be useful for screening and management of patients with cancer.

Clinical implications of genomic alterations in the tumour and circulation of pancreatic cancer patients.

Pancreatic adenocarcinoma has the worst mortality of any solid cancer. In this study, to evaluate the clinical implications of genomic alterations in this tumour type, we perform whole-exome analyses of 24 tumours, targeted genomic analyses of 77 tumours, and use non-invasive approaches to examine tumour-specific mutations in the circulation of these patients. These analyses reveal somatic mutations in chromatin-regulating genes MLL, MLL2, MLL3 and ARID1A in 20% of patients that are associated with improved survival. We observe alterations in genes with potential therapeutic utility in over a third of cases. Liquid biopsy analyses demonstrate that 43% of patients with localized disease have detectable circulating tumour DNA (ctDNA) at diagnosis. Detection of ctDNA after resection predicts clinical relapse and poor outcome, with recurrence by ctDNA detected 6.5 months earlier than with CT imaging. These observations provide genetic predictors of outcome in pancreatic cancer and have implications for new avenues of therapeutic intervention.

Personalized genomic analyses for cancer mutation discovery and interpretation.

Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer-predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false-positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients.

Increased Expression of Phosphorylated FADD in Anaplastic Large Cell and Other T-Cell Lymphomas.

FAS-associated protein with death domain (FADD) is a major adaptor protein involved in extrinsic apoptosis, embryogenesis, and lymphocyte homeostasis. Although abnormalities of the FADD/death receptor apoptotic pathways have been established in tumorigenesis, fewer studies have analyzed the expression and role of phosphorylated FADD (pFADD). Our identification of FADD as a lymphoma-associated autoantigen in T-cell lymphoma patients raises the possibility that pFADD, with its correlation with cell cycle, may possess role(s) in human T-cell lymphoma development. This immunohistochemical study investigated pFADD protein expression in a range of normal tissues and lymphomas, particularly T-cell lymphomas that require improved therapies. Whereas pFADD was expressed only in scattered normal T cells, it was detected at high levels in T-cell lymphomas (eg, 84% anaplastic large cell lymphoma and 65% peripheral T cell lymphomas, not otherwise specified). The increased expression of pFADD supports further study of its clinical relevance and role in lymphomagenesis, highlighting phosphorylation of FADD as a potential therapeutic target.

RNASET2--an autoantigen in anaplastic large cell lymphoma identified by protein array analysis.

Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted.

MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-ß signalling pathway.

Transforming growth factor-ß (TGF-ß) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-ß non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-ß pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-ß signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-ß non-canonical signaling by miR-335, which in turn is downregulated by MYCN.

Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase A oncogene.

BACKGROUND: Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. RESULTS: As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. CONCLUSIONS: PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

Co-localization of the oncogenic transcription factor MYCN and the DNA methyl binding protein MeCP2 at genomic sites in neuroblastoma.

BACKGROUND: MYCN is a transcription factor that is expressed during the development of the neural crest and its dysregulation plays a major role in the pathogenesis of pediatric cancers such as neuroblastoma, medulloblastoma and rhabdomyosarcoma. MeCP2 is a CpG methyl binding protein which has been associated with a number of cancers and developmental disorders, particularly Rett syndrome. METHODS AND FINDINGS: Using an integrative global genomics approach involving chromatin immunoprecipitation applied to microarrays, we have determined that MYCN and MeCP2 co-localize to gene promoter regions, as well as inter/intragenic sites, within the neuroblastoma genome (MYCN amplified Kelly cells) at high frequency (70.2% of MYCN sites were also positive for MeCP2). Intriguingly, the frequency of co-localization was significantly less at promoter regions exhibiting substantial hypermethylation (8.7%), as determined by methylated DNA immunoprecipitation (MeDIP) applied to the same microarrays. Co-immunoprecipitation of MYCN using an anti-MeCP2 antibody indicated that a MYCN/MeCP2 interaction occurs at protein level. mRNA expression profiling revealed that the median expression of genes with promoters bound by MYCN was significantly higher than for genes bound by MeCP2, and that genes bound by both proteins had intermediate expression. Pathway analysis was carried out for genes bound by MYCN, MeCP2 or MYCN/MeCP2, revealing higher order functions. CONCLUSIONS: Our results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency, and that the patterns of binding of these proteins can be associated with significant differences in transcriptional activity. Although it is not yet known if this interaction contributes to neuroblastoma disease pathogenesis, it is intriguing that the interaction occurs at the promoter regions of several genes important for the development of neuroblastoma, including ALK, AURKA and BDNF.

Dissection of the oncogenic MYCN transcriptional network reveals a large set of clinically relevant cell cycle genes as drivers of neuroblastoma tumorigenesis.

Amplification of the oncogenic transcription factor MYCN plays a major role in the pathogenesis of several pediatric cancers, including neuroblastoma, medulloblastoma, and rhabodomyosarcoma. For neuroblastoma, MYCN amplification is the most powerful genetic predictor of poor patient survival, yet the mechanism by which MYCN drives tumorigenesis is only partially understood. To gain an insight into the distribution of MYCN binding and to identify clinically relevant MYCN target genes, we performed an integrated analysis of MYCN ChIP-chip and mRNA expression using the MYCN repressible SHEP-21N neuroblastoma cell line. We hypothesized that genes exclusively MYCN bound in SHEP-21N cells over-expressing MYCN would be enriched for direct targets which contribute to the process of disease progression. Integrated analysis revealed that MYCN drives tumorigenesis predominantly as a positive regulator of target gene transcription. A high proportion of genes (24%) that are MYCN bound and up-regulated in the SHEP-21N model are significantly associated with poor overall patient survival (OS) in a set of 88 tumors. In contrast, the proportion of genes down-regulated when bound by MYCN in the SHEP-21N model and which are significantly associated with poor overall patient survival when under-expressed in primary tumors was significantly lower (5%). Gene ontology analysis determined a highly statistically significant enrichment for cell cycle related genes within the over-expressed MYCN target group which were also associated with poor OS. We conclude that the over-expression of MYCN leads to aberrant binding and over-expression of genes associated with cell cycle regulation which are significantly correlated with poor OS and MYCN amplification.

MicroRNA mediates DNA demethylation events triggered by retinoic acid during neuroblastoma cell differentiation.

Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regimen for patients with high-risk disease, and a similar derivative, all-trans-retinoic acid (ATRA), causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA-induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. Four hundred and two gene promoters became demethylated, whereas 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were overexpressed by >2-fold, whereas 13 of the hypermethylated genes were underexpressed. Gene ontology analysis indicated that demethylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we show the downregulation of methyltransferases, DNMT1 and DNMT3B, along with the upregulation of endogenous microRNAs targeting them. Ectopic overexpression of miR-152, targeting DNMT1, also negatively affected cell invasiveness and anchorage-independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA demethylation changes contribute to the process of ATRA-induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation.

Protein interactions with the platelet integrin alpha(IIb) regulatory motif.

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin alpha-subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet-specific integrin alpha(IIb)beta(3). We employ peptide-affinity chromatography followed-up with LC-MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR-peptide. Of these, seven have been reported in the PPI literature as interactors with integrin alpha-subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin-tagged alpha-integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide-affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.

MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2.

BACKGROUND: Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. RESULTS: We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. CONCLUSIONS: MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

Constitutively overexpressed 21 kDa protein in Hodgkin lymphoma and aggressive non-Hodgkin lymphomas identified as cytochrome B5b (CYB5B).

BACKGROUND: We have previously reported a novel constitutively overexpressed 21 kDa protein in Hodgkin Lymphoma (HL) and aggressive Non-Hodgkin Lymphomas (NHL). The objective of the current study was to 1) identify this protein using two independent methods, 2) study the expression of the protein and its encoding mRNA in reactive lymph nodes, normal lymphocytes and CD34+ bone marrow precursor cells, 3) analyse patterns of expression of the protein in tissue microarrays assembled from a large number of diagnostic clinical biopsies from patients with HL, and 4) determine the copy number variation and mutation status of the encoding gene in HL cell lines. RESULTS: Peptide sequencing by LC-MS/MS and protein identification by protein array screening identified a single protein, CYB5B. No mutations were detected in the CYB5B gene in HL cell lines. Quantitative PCR showed CYB5B gene expression was increased in HL and NHL cell lines. Array CGH using a submegabase resolution tiling array revealed gains in the CYB5B locus in HL cell lines KMH2 and L428. Membrane expression was seen in Reed-Sternberg cells in clinical biopsies from patients with HL but not in reactive lymph nodes. Bone marrow CD34+ precursor cells were CYB5B negative on the cell surface. RT-PCR assays of RNA extracted from T and B cell enriched fractions obtained from normal peripheral blood mononuclear cells, reactive lymph nodes, tonsils and normal bone marrow samples showed no evidence of increased mRNA levels of CYB5B in comparison to housekeeping gene GAPDH. CONCLUSIONS: The 21 kDa protein overexpressed in HL and aggressive NHL is identical to CYB5B. CYB5B gene expression is increased in a subset of HL and NHL cell lines tested. This is associated with CYB5B gene amplification in HL cell lines KMH2 and L428. CYB5B may be a potential target for antibody-based therapy of HL and aggressive NHL as although cytoplasmic expression is present in reactive lymphocytes, it is not expressed on the cell surface of non-neoplastic lymphocytes or bone marrow precursor cells.

Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation.

BACKGROUND: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. METHODOLOGY: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. CONCLUSION: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the activity of individual genes, and that of a mediator of global chromatin structure.

Widespread dysregulation of MiRNAs by MYCN amplification and chromosomal imbalances in neuroblastoma: association of miRNA expression with survival.

MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.

Protein arrays as tools for serum autoantibody marker discovery in cancer.

Protein array technology has begun to play a significant role in the study of protein-protein interactions and in the identification of antigenic targets of serum autoantibodies in a variety of autoimmune disorders. More recently, this technology has been applied to the identification of autoantibody signatures in cancer. The identification of tumour-associated antigens (TAAs) recognised by the patient's immune response represents an exciting approach to identify novel diagnostic cancer biomarkers and may contribute towards a better understanding of the molecular mechanisms involved. Circulating autoantibodies have not only been used to identify TAAs as diagnostic/prognostic markers and potential therapeutic targets, they also represent excellent biomarkers for the early detection of tumours and potential markers for monitoring the efficacy of treatment. Protein array technology offers the ability to screen the humoral immune response in cancer against thousands of proteins in a high throughput technique, thus readily identifying new panels of TAAs. Such an approach should not only aid in improved diagnostics, but has already contributed to the identification of complex autoantibody signatures that may represent disease subgroups, early diagnostics and facilitated the analysis of vaccine trials.

Downregulation of p21(waf/cip-1) mediates apoptosis of human hepatocellular carcinoma cells in response to interferon-gamma.

There is no effective treatment for advanced hepatocellular carcinoma (HCC). We therefore explored the molecular mechanisms of interferon-gamma (IFN-gamma)-mediated growth regulation in human HCC cell lines. IFN-gamma receptor expression, signal transduction, and regulation of effectors were examined by RT-PCR, immunoprecipitation, immunoblotting, and reporter gene assays. Growth and apoptosis were determined based on cell numbers, cell cycle analyses, kinase assays, DNA fragmentation, and PARP cleavage. HCC cell lines express functionally intact IFN-gamma receptors and downstream effectors. IFN-gamma profoundly inhibited growth of HCC cells via two different mechanisms: inhibition of G1 cell cycle progression and induction of apoptosis. Analyses in SK-Hep-1 cells revealed a deficient cyclin D induction in IFN-gamma-treated cells, resulting in reduced activity of CDK4 and CDK2 kinases and pRB hypophosphorylation. In contrast, apoptosis prevailed in IFN-gamma-treated HepG2 cultures. A survey of apoptosis relevant IFN-gamma effectors including IRF-1, caspase-1, caspase-3, and p21(waf/cip-1) documented a dramatic transcriptional downregulation of p21(waf/cip-1) exclusively in apoptosis-susceptible HepG2 cells. Reconstitution of p21(waf/cip-1) rescued HepG2 cells from IFN-gamma-induced apoptosis, indicating that p21(waf/cip-1) reduction was required for apoptosis execution. Inversely, downregulation of p21(waf/cip-1) sensitized SK-Hep-1 cells to IFN-gamma-induced apoptosis. Thus, downregulation of p21(waf/cip-1) in HCC cells functions as a novel, critical determinant of alternative growth inhibitory pathways in response to IFN-gamma.