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Supplementary Materials and Methods; Supplementary Tables S1-S3.

Purpose: Blood-based surrogate markers would be attractive biomarkers for early detection, diagnosis, prognosis, and prediction of therapeutic outcome in cancer. Disease-associated gene expression signatures in peripheral blood mononuclear cells (PBMC) have been described for several cancer types. However, RNA-stabilized whole blood–based technologies would be clinically more applicable and robust. We evaluated the applicability of whole blood–based gene expression profiling for the detection of non–small cell lung cancer (NSCLC).Experimental Design: Expression profiles were generated from PAXgene-stabilized blood samples from three independent groups consisting of NSCLC cases and controls (n = 77, 54, and 102), using the Illumina WG6-VS2 system.Results: Several genes are consistently differentially expressed in whole blood of NSCLC patients and controls. These expression profiles were used to build a diagnostic classifier for NSCLC, which was validated in an independent validation set of NSCLC patients (stages I–IV) and hospital-based controls. The area under the receiver operator curve was calculated to be 0.824 (P < 0.001). In a further independent dataset of stage I NSCLC patients and healthy controls the AUC was 0.977 (P < 0.001). Specificity of the classifier was validated by permutation analysis in both validation cohorts. Genes within the classifier are enriched in immune-associated genes and show specificity for NSCLC.Conclusions: Our results show that gene expression profiles of whole blood allow for detection of manifest NSCLC. These results prompt further development of gene expression–based biomarker tests in peripheral blood for the diagnosis and early detection of NSCLC. Clin Cancer Res; 17(10); 3360–7. ©2011 AACR.

Many tumors, including Hodgkin's lymphoma, are associated with decreased cellular immunity and elevated levels of prostaglandin E2 (PGE2), a known inhibitor of CD4+ T cell activation, suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE2 might have on primary human CD4+ T cells, we used a whole genome-based transcriptional approach and show that PGE2 severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE2 at an early step of T cell receptor signaling: indeed, PGE2 stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE2-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence, PGE2-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27kip1. Most importantly, CD4+ T cells in Hodgkin's lymphoma show similar regulation of genes that were altered in vitro by PGE2 in T cells from healthy individuals. These data strongly suggest that PGE2 is an important factor leading to CD4+ T cell impairment observed in Hodgkin's lymphoma. (Cancer Res 2006; 66(2): 1114-22)

Supplementary Figure Legends 1-12, Table Legends 1-4, Results from Cancer Vaccine Enhanced, Non–Tumor-Reactive CD8+ T Cells Exhibit a Distinct Molecular Program Associated with “Division Arrest Anergy”

Supplementary Table 2 from Prostaglandin E2 Impairs CD4+ T Cell Activation by Inhibition of lck: Implications in Hodgkin's Lymphoma

Supplementary Table 1 from Prostaglandin E2 Impairs CD4+ T Cell Activation by Inhibition of lck: Implications in Hodgkin's Lymphoma

Supplementary Figures 1-12 from Cancer Vaccine Enhanced, Non–Tumor-Reactive CD8+ T Cells Exhibit a Distinct Molecular Program Associated with “Division Arrest Anergy”

The function of the IgM B-cell receptor (BCR) is dependent on intact signaling of the co-receptors Igα and Igβ, both of which contain a cytoplasmic tail bearing an immunoreceptor tyrosine-based activation motif. We have previously demonstrated that the cytoplasmic tail of the IgG1 BCR can partially compensate for the loss of the signaling moiety of Igα. Here, we show that unlike Igα, Igβ signaling is indispensable for the development and function of IgG1-expressing B cells. Deletion of the cytoplasmic signaling tail of Igβ compromised the survival and proliferation not only of IgM(+) B cells but also of IgG1-expressing B cells. In the absence of the signaling tail of Igβ, the transcription levels of the antiapoptotic gene bcl-xl and the cell-cycle gene ccnd2 were reduced, consistent with the observed defects in survival and proliferation. These results demonstrate functional differences between Igα and Igβ in the transduction of IgG1 BCR signal.

Purpose: Blood-based surrogate markers would be attractive biomarkers for early detection, diagnosis, prognosis, and prediction of therapeutic outcome in cancer. Disease-associated gene expression signatures in peripheral blood mononuclear cells (PBMC) have been described for several cancer types. However, RNA-stabilized whole blood–based technologies would be clinically more applicable and robust. We evaluated the applicability of whole blood–based gene expression profiling for the detection of non–small cell lung cancer (NSCLC). Experimental Design: Expression profiles were generated from PAXgene-stabilized blood samples from three independent groups consisting of NSCLC cases and controls (n = 77, 54, and 102), using the Illumina WG6-VS2 system. Results: Several genes are consistently differentially expressed in whole blood of NSCLC patients and controls. These expression profiles were used to build a diagnostic classifier for NSCLC, which was validated in an independent validation set of NSCLC patients (stages I–IV) and hospital-based controls. The area under the receiver operator curve was calculated to be 0.824 (P < 0.001). In a further independent dataset of stage I NSCLC patients and healthy controls the AUC was 0.977 (P < 0.001). Specificity of the classifier was validated by permutation analysis in both validation cohorts. Genes within the classifier are enriched in immune-associated genes and show specificity for NSCLC. Conclusions: Our results show that gene expression profiles of whole blood allow for detection of manifest NSCLC. These results prompt further development of gene expression–based biomarker tests in peripheral blood for the diagnosis and early detection of NSCLC. Clin Cancer Res; 17(10); 3360–7. ©2011 AACR.

Blood-based mRNA expression profiling has already become an important issue in clinical applications. More recently, the characterization of the small RNA transcriptome offers additional avenues for diagnostic approaches. However, when applying miRNA expression profiling in routine clinical settings, the method of RNA preservation and the manner of RNA extraction as well as the reliability of the miRNA profiling procedure have to be carefully considered. Here we evaluate a recently introduced bead array–based technology as a robust method for the generation of blood-based human miRNA expression profiles. Importantly the comparison of different RNA extraction strategies resulted in dissimilar profiles depending on the RNA extraction method as well as on the underlying source. Expression profiles obtained from peripheral mononuclear cells (PBMCs) substantially differed from those of whole blood samples, whereby both sources per se yielded reproducible and reliable results. Expression profiles were also distinct when using either fresh or frozen PBMCs. Moreover RNA size fractioning resulted in discriminative miRNA expression profiles compared with total RNA based profiles. This study outlines important steps toward the establishment of a robust strategy for blood-based miRNA profiling and provides a reliable strategy for its implementation in routine handling for diagnostic purposes.

Dendritic cells are able to take up exogenous Ags and present Ag-derived peptides on MHC class I molecules, a process termed cross-presentation. The mannose receptor (MR), an endocytic receptor expressed on a variety of APCs, has been demonstrated to target soluble Ags exclusively toward cross-presentation. In this study, we investigated the role of the murine nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated transcription factor with immunomodulatory properties, in MR-mediated endocytosis and cross-presentation of the model Ag OVA. We could demonstrate both in vitro and in vivo that activation of PPARγ resulted in increased MR expression, which in consequence led to enhanced MR-mediated endocytosis and elevated cross-presentation of soluble OVA. Concomitantly, activation of PPARγ in dendritic cells induced up-regulation of the coinhibitory molecule B7H1, which, despite enhanced cross-presentation, caused an impaired activation of naive OVA-specific CD8+ T cells and the induction of T cell tolerance. These data provide a mechanistic basis for the immunomodulatory action of PPARγ which might open new possibilities in the development of therapeutic approaches aimed at the control of excessive immune responses, e.g., in T cell-mediated autoimmunity.

Tumorerkrankungen stellen in Deutschland nach wie vor die zweithaufigste Tordesursache dar. Neben der Entwicklung wirksamer primarer und sekundarer Praventionsmasnahmen sind vor allem Methoden zur Verbesserung der Diagnose und Subklassifikation sowie zur Prognoseeinschatzung der einzelnen Tumorentitaten dazu geeignet, die krebsbedingte Letalitat langfristig zu senken. Die klassische Diagnose eines Tumors stutzt sich vielfach auf seine histomorphologische Charakterisierung, wohingegen die prognostische Einschatzung zusatzlich durch das Stadium der Erkrankung bei Diagnose bestimmt wird. Nach der Etablierung von Hochdurchsatzmethoden zur Erstellung von Genexpressionsprofilen von Tumorbiopsien wurde im vergangenen Jahrzehnt untersucht, ob die molekulare Charakterisierung von Tumoren die diagnostische Sicherheit zusatzlich erhohen und die Einschatzung der Prognose verbessern kann. Hierzu wurden vor allem Studien beim Mamma-, Lungen- und Kolonkarzinom durchgefuhrt mit dem Ziel, in der adjuvanten Situation bei Vorliegen eines fruhen Stadiums feststellen zu konnen, welche Patientenkollektive tatsachlich von einer Chemotherapie profitieren wurden. Ferner wurden Genexpressionsprofile dazu genutzt, Tumoren mit unklarem Primarius ihrem Ursprungsgewebe zuzuordnen. Daruber hinaus wurde mittels Genexpressionsanalyse eine erganzende molekulare Klassifikation sowohl von B-Zell-Non-Hodgkin-Lymphomen als auch von lymphatischen und myeloischen Leukamien ermoglicht, wodurch moglicherweise sowohl die Diagnostik als auch die Einschatzung der Prognose dieser Erkrankungen langfristig verbessert werden konnen. Obwohl mittlerweile zahlreiche Signaturen und Pradiktoren zu einzelnen Neoplasien veroffentlicht wurden, fand in Deutschland bisher keiner der Tests Eingang in die leitlinienbasierte Diagnostik und Therapie einer malignen Erkrankung. Dies ist vor allem darauf zuruckzufuhren, dass die vielversprechenden Ergebnisse zunachst in grosen prospektiven klinischen Studien evaluiert werden mussen. Erst nach Abschluss dieser Studien wird zu entscheiden sein, ob und welche Testverfahren tatsachlich geeignet sind, den Verlauf einer bestimmten Tumorerkrankung signifikant zu verbessern.

In recent years, the usage of high-throughput technologies in the fields of genomics, transcriptomics, proteomics and metabolomics for biomarker discovery has expanded enormously. Biomarkers can be applied for many purposes, including diagnosis, prognosis, staging and selecting appropriate patient therapy. In addition, biomarkers can provide information on disease mechanism or progression. Biomarker development for clinical application encompasses phases for their discovery and characterization, assay development and, finally, implementation using automated platforms employed in clinical laboratories. However, translation from bench to bedside outside a research-oriented environment has proven to be more difficult. This is reflected by only few new biomarkers being integrated into clinical application in the last years. This article reviews currently used high-throughput technologies for the identification of biomarkers, as well as present approaches to increase the percentage of biomarkers that pass the barriers for clinical application.

Immune tolerance is a central mechanism counteracting tumor-specific immunity and preventing effective anticancer immunotherapy. Induction of tolerance requires a specific environment in which tolerogenic dendritic cells (DCs) play an essential role deviating the immune response away from effective immunity. It was recently shown that maturation of DCs in the presence of PGE2 results in upregulation of indoleamine 2,3-dioxygenase (IDO) providing a potential mechanism for the development of DC-mediated Tcell tolerance. Here, we extend these findings, demonstrating a concomitant induction of IDO and secretion of soluble CD25 after DC maturation in the presence of PGE2. While maturation of DCs induced IDO expression on transcriptional level, only integration of PGE2 signaling led to up-regulation of functional IDO protein as well as significant expression of cell-surface and soluble CD25 protein. As a consequence, T-cell proliferation and cytokine production were significantly inhibited, which was mediated mainly by IDO-induced tryptophan depletion. Of importance, we demonstrate that different carcinoma entities associated with elevated levels of PGE2 coexpress CD25 and IDO in peritumoral dendritic cells, suggesting that PGE2 might influence IDO expression in human DCs in the tumor environment. We therefore suggest PGE2 to be a mediator of early events during induction of immune tolerance in cancer. (Blood. 2006;108:228-237)

Many tumors, including Hodgkin's lymphoma, are associated with decreased cellular immunity and elevated levels of prostaglandin E2 (PGE2), a known inhibitor of CD4+ T cell activation, suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE2 might have on primary human CD4+ T cells, we used a whole genome-based transcriptional approach and show that PGE2 severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE2 at an early step of T cell receptor signaling: indeed, PGE2 stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE2-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence, PGE2-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27kip1. Most importantly, CD4+ T cells in Hodgkin's lymphoma show similar regulation of genes that were altered in vitro by PGE2 in T cells from healthy individuals. These data strongly suggest that PGE2 is an important factor leading to CD4+ T cell impairment observed in Hodgkin's lymphoma. (Cancer Res 2006; 66(2): 1114-22)

The onset and structure of compositional convection in a rotating system are investigated experimentally. A vertically oriented cylindrical annulus filled with NH 4 Cl-H 2 O solution is cooled from the bottom and can be rotated about its axis at rates ranging up to 10.5 rad s -1 , corresponding to Ekman numbers down to 7.5 x 10 -6 . The Coriolis force has a strong effect on the structure of plumes above the mush-liquid interface. Helical motion of the conduit, which is weakly developed in the non-rotating case, is amplified by Coriolis forces that twist the plume conduits to lie nearly horizontally. This results in secondary plumes (or blobs) that rise from the sub-horizontal primary plume conduits. This new instability could be an efficient mechanism for producing small-scale flow in the form of buoyant blobs that ascend through the polar regions of the outer core.

In recent years an increase of functional CD4+CD25+regulatory T cells (Tregcells) has been established for patients with solid tumors, acute leukemias, and lymphomas. We have reported an expanded pool of CD4+CD25highTregcells in patients with chronic lymphatic leukemia (CLL), multiple myeloma (MM) as well as its premalignant precursor monoclonal gammopathy of undetermined significance (MGUS). In healthy individuals, low-level expression of CD127 on T cells in addition to the expression of FOXP3 has been associated with Tregcells. Here, we demonstrate that the expanded FOXP3+T-cell population in patients with colorectal cancer, CLL, MGUS, MM, follicular lymphoma, and Hodgkin's disease are exclusively CD127lowTregcells and were strongly suppressive. A significant portion of CD127lowFOXP3+Tregcells expressed only low levels of CD25 suggesting that the previously reported expansion of CD25+Tregcells underestimates the true expansion. The assessment of CCR7 and CD45RA expression on the expanded CD4+CD127lowFOXP3+Tregcells revealed an increase of both naïve as well as central and effector memory Tregcells in peripheral blood. Our data strongly support superiority of combined CD127 and FOXP3 analysis in comparison to CD25 and FOXP3 assessment for further quantification of Tregcells in malignant diseases.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

In 1999, Golub et al. proposed for the first time microarray-based transcriptional profiling to be used as a new technology for the differential diagnosis of acute myeloid leukemias and acute lymphocytic leukemias. This very preliminary study sparked great enthusiasm beyond the leukemias. Over the last 10 years, numerous studies addressed the use of gene expression profiling of peripheral blood from patients with malignancies, infectious diseases, autoimmunity and even cardiovascular diseases. Despite this great effort, no single test has yet been established using microarray-based transcriptional profiling of peripheral blood. Here we highlight the advances in the field of blood transcriptomics during the last 10 years and also critically discuss the issues that need to be resolved before blood transcriptomics will become part of daily diagnostics in the leukemias, as well as in other diseases showing involvement of peripheral blood.

Immune-mediated tumor rejection relies on fully functional T-cell responses and neutralization of an adverse tumor microenvironment. In clinical trials, we detected peptide-specific but non–tumor-reactive and therefore not fully functional CD8+ T cells post-vaccination against tumor antigens. Understanding the molecular mechanisms behind nontumor reactivity will be a prerequisite to overcome this CD8+ T-cell deviation. We report that these non–tumor-reactive CD8+ T cells are characterized by a molecular program associated with hallmarks of “division arrest anergy.” Non–tumor-reactive CD8+ T cells are characterized by coexpression of CD7, CD25, and CD69 as well as elevated levels of lckp505 and p27kip1. In vivo quantification revealed high prevalence of non–tumor-reactive CD8+ T cells with increased levels during cancer vaccination. Furthermore, their presence was associated with a trend toward shorter survival. Dynamics and frequencies of non–target-reactive CD8+ T cells need to be further addressed in context of therapeutic vaccine development in cancer, chronic infections, and autoimmune diseases. [Cancer Res 2009;69(10):4346–54]

Based on studies in knockout mice, several inhibitory factors such as TGFβ, IL-10, or CTLA-4 have been implicated as gate keepers of adaptive immune responses. Lack of these inhibitory molecules leads to massive inflammatory responses mainly mediated by activated T cells. In humans, the integration of these inhibitory signals for keeping T cells at a resting state is less well understood. To elucidate this regulatory network, we assessed early genome-wide transcriptional changes during serum deprivation in human mature CD4+ T cells. The most striking observation was a “TGFβ loss signature” defined by down-regulation of many known TGFβ target genes. Moreover, numerous novel TGFβ target genes were identified that are under the suppressive control of TGFβ. Expression of these genes was up-regulated once TGFβ signaling was lost during serum deprivation and again suppressed upon TGFβ reconstitution. Constitutive TGFβ signaling was corroborated by demonstrating phosphorylated SMAD2/3 in resting human CD4+ T cells in situ, which were dephosphorylated during serum deprivation and rephosphorylated by minute amounts of TGFβ. Loss of TGFβ signaling was particularly important for T cell proliferation induced by low-level TCR and costimulatory signals. We suggest TGFβ to be the most prominent factor actively keeping human CD4+ T cells at a resting state.

We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) gamma1 or mu heavy chains. Progenitor cells expressing gamma1 chains from the beginning generate a peripheral B cell compartment of normal size with all subsets, but a partial block is seen at the pro- to pre-B cell transition. Accordingly, gamma1-driven B cell development is disfavored in competition with developing B cells expressing a wild-type (WT) IgH locus. However, the mutant B cells display a long half-life and accumulate in the mature B cell compartment, and even though partial truncation of the Ig alpha cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca(2+) response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca(2+) response was also observed in mature B cells that had been switched from IgM to IgG1 expression in vivo. Collectively, these results suggest that the gamma1 chain can exert a unique signaling function that can partially replace that of the Ig alpha/beta heterodimer in B cell maintenance and may contribute to memory B cell physiology.

In solid tumors, leukemias, and lymphomas, increased frequencies of functional CD4+CD25high regulatory T cells (Treg cells) have been previously demonstrated. In healthy individuals, Treg cells consist not only of memory but also of naive T cells, which can undergo peripheral expansion and are characterized by a relative enrichment for autoreactive T-cell receptors. Here, we demonstrate in patients with premalignant monoclonal gammopathy of undetermined significance and patients with multiple myeloma that functional FoxP3+ Treg cells of naive, central, and effector memory phenotype as determined by CCR7 and CD45RA expression are significantly expanded. Low frequencies of T-cell receptor excision circles in naive Treg cells in both healthy controls and multiple myeloma patients point to peripheral expansion as the prominent mechanism of increased frequencies of naive Treg cells in these cancer patients. These findings strongly suggest that the increase of functional Treg cells in cancer patients is a response to the process of malignant transformation.

Regulatory T cells (T(reg) cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of T(reg) cells was established. In IL-2 treated cancer patients a further T(reg)-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional T(reg) cells of a naïve phenotype--as determined by CCR7 and CD45RA expression--are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve T(reg)-cell pool. Higher frequencies of T-cell receptor excision circles in naïve T(reg) cells indicate IL-2 dependent thymic generation of naïve T(reg) cells as a mechanism leading to increased frequencies of T(reg) cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine T(reg) cells after IL-2 administration. These results point to a more complex regulation of T(reg) cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve T(reg) cells.

Background Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells. Results Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes. Conclusions These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.