Development and validation of decision rules to guide frequency of monitoring CD4 cell count in HIV-1 infection before starting antiretroviral therapy

Background Although CD4 cell count monitoring is used to decide when to start antiretroviral therapy in patients with HIV-1 infection, there are no evidence-based recommendations regarding its optimal frequency. It is common practice to monitor every 3 to 6 months, often coupled with viral load monitoring. We developed rules to guide frequency of CD4 cell count monitoring in HIV infection before starting antiretroviral therapy, which we validated retrospectively in patients from the Swiss HIV Cohort Study. Methodology/Principal Findings We built up two prediction rules (“Snap-shot rule” for a single sample and “Track-shot rule” for multiple determinations) based on a systematic review of published longitudinal analyses of CD4 cell count trajectories. We applied the rules in 2608 untreated patients to classify their 18 061 CD4 counts as either justifiable or superfluous, according to their prior ≥5% or <5% chance of meeting predetermined thresholds for starting treatment. The percentage of measurements that both rules falsely deemed superfluous never exceeded 5%. Superfluous CD4 determinations represented 4%, 11%, and 39% of all actual determinations for treatment thresholds of 500, 350, and 200×106/L, respectively. The Track-shot rule was only marginally superior to the Snap-shot rule. Both rules lose usefulness for CD4 counts coming near to treatment threshold. Conclusions/Significance Frequent CD4 count monitoring of patients with CD4 counts well above the threshold for initiating therapy is unlikely to identify patients who require therapy. It appears sufficient to measure CD4 cell count 1 year after a count >650 for a threshold of 200, >900 for 350, or >1150 for 500×106/L, respectively. When CD4 counts fall below these limits, increased monitoring frequency becomes advisable. These rules offer guidance for efficient CD4 monitoring, particularly in resource-limited settings.

Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of morphology-based approaches. Copyright © 2012 John Wiley & Sons, Ltd.

Modeling of red blood cell life-spans in hematologically normal populations

Despite the impact of red blood cell (RBC) Life-spans in some disease areas such as diabetes or anemia of chronic kidney disease, there is no consensus on how to quantitatively best describe the process. Several models have been proposed to explain the elimination process of RBCs: random destruction process, homogeneous life-span model, or a series of 4-transit compartment model. The aim of this work was to explore the different models that have been proposed in literature, and modifications to those. The impact of choosing the right model on future outcomes prediction--in the above mentioned areas--was also investigated. Both data from indirect (clinical data) and direct life-span measurement (biotin-labeled data) methods were analyzed using non-linear mixed effects models. Analysis showed that: (1) predictions from non-steady state data will depend on the RBC model chosen; (2) the transit compartment model, which considers variation in life-span in the RBC population, better describes RBC survival data than the random destruction or homogenous life-span models; and (3) the additional incorporation of random destruction patterns, although improving the description of the RBC survival data, does not appear to provide a marked improvement when describing clinical data.

Post-translational signal peptide cleavage controls differential epitope recognition in the QP-rich domain of recombinant Theileria parva PIM

The presence of the schizont stage of the obligate intracellular parasites Theileria parva or T. annulata in the cytoplasm of an infected leukocyte results in host cell transformation via a mechanism that has not yet been elucidated. Proteins, secreted by the schizont, or expressed on its surface, are of interest as they can interact with host cell molecules that regulate host cell proliferation and/or survival. The major schizont surface protein is the polymorphic immunodominant molecule, PIM, which contains a large glutamine- and proline-rich domain (QP-rd) that protrudes into the host cell cytoplasm. Analyzing QP-rd generated by in vitro transcription/translation, we found that the signal peptide was efficiently cleaved post-translationally upon addition of T cell lysate or canine pancreatic microsomes, whereas signal peptide cleavage of a control protein only occurred cotranslationally and in the presence of microsomal membranes. The QP-rd of PIM migrated anomalously in SDS-PAGE and removal of the 19 amino acids corresponding to the predicted signal peptide caused a decrease in apparent molecular mass of 24kDa. The molecule was analyzed using monoclonal antibodies that recognize a set of previously defined PIM epitopes. Depending on the presence or the absence of the signal peptide, two conformational states could be demonstrated that are differentially recognized, with N-terminal epitopes becoming readily accessible upon signal peptide removal, and C-terminal epitopes becoming masked. Similar observations were made when the QP-rd of PIM was expressed in bacteria. Our observations could also be of relevance to other schizont proteins. A recent analysis of the proteomes of T. parva and T. annulata revealed the presence of a large family of potentially secreted proteins, characterized by the presence of large stretches of amino acids that are also particularly rich in QP-residues.

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

Progenitor cell recruitment during individualized high-flow, very-large-volume apheresis for autologous transplantation improves collection efficiency

BACKGROUND: Individual adaptation of processed patient's blood volume (PBV) should reduce number and/or duration of autologous peripheral blood progenitor cell (PBPC) collections. STUDY DESIGN AND METHODS: The durations of leukapheresis procedures were adapted by means of an interim analysis of harvested CD34+ cells to obtain the intended yield of CD34+ within as few and/or short as possible leukapheresis procedures. Absolute efficiency (AE; CD34+/kg body weight) and relative efficiency (RE; total CD34+ yield of single apheresis/total number of preapheresis CD34+) were calculated, assuming an intraapheresis recruitment if RE was greater than 1, and a yield prediction models for adults was generated. RESULTS: A total of 196 adults required a total of 266 PBPC collections. The median AE was 7.99 x 10(6), and the median RE was 1.76. The prediction model for AE showed a satisfactory predictive value for preapheresis CD34+ only. The prediction model for RE also showed a low predictive value (R2 = 0.36). Twenty-eight children underwent 44 PBPC collections. The median AE was 12.13 x 10(6), and the median RE was 1.62. Major complications comprised bleeding episodes related to central venous catheters (n = 4) and severe thrombocytopenia of less than 10 x 10(9) per L (n = 16). CONCLUSION: A CD34+ interim analysis is a suitable tool for individual adaptation of the duration of leukapheresis. During leukapheresis, a substantial recruitment of CD34+ was observed, resulting in a RE of greater than 1 in more than 75 percent of patients. The upper limit of processed PBV showing an intraapheresis CD34+ recruitment is higher than in a standard large-volume leukapheresis. Therefore, a reduction of individually needed PBPC collections by means of a further escalation of the processed PBV seems possible.

Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura

BACKGROUND: The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. METHODS: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. RESULTS: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. CONCLUSION: Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.

Characterization of thermal and mechanical properties of polypropylene-based composites for fuel cell bipolar plates and development of educational tools in hydrogen and fuel cell technologies

In this project we developed conductive thermoplastic resins by adding varying amounts of three different carbon fillers: carbon black (CB), synthetic graphite (SG) and multi-walled carbon nanotubes (CNT) to a polypropylene matrix for application as fuel cell bipolar plates. This component of fuel cells provides mechanical support to the stack, circulates the gases that participate in the electrochemical reaction within the fuel cell and allows for removal of the excess heat from the system. The materials fabricated in this work were tested to determine their mechanical and thermal properties. These materials were produced by adding varying amounts of single carbon fillers to a polypropylene matrix (2.5 to 15 wt.% Ketjenblack EC-600 JD carbon black, 10 to 80 wt.% Asbury Carbon's Thermocarb TC-300 synthetic graphite, and 2.5 to 15 wt.% of Hyperion Catalysis International's FIBRILTM multi-walled carbon nanotubes) In addition, composite materials containing combinations of these three fillers were produced. The thermal conductivity results showed an increase in both through-plane and in-plane thermal conductivities, with the largest increase observed for synthetic graphite. The Department of Energy (DOE) had previously set a thermal conductivity goal of 20 W/m·K, which was surpassed by formulations containing 75 wt.% and 80 wt.% SG, yielding in-plane thermal conductivity values of 24.4 W/m·K and 33.6 W/m·K, respectively. In addition, composites containing 2.5 wt.% CB, 65 wt.% SG, and 6 wt.% CNT in PP had an in–plane thermal conductivity of 37 W/m·K. Flexural and tensile tests were conducted. All composite formulations exceeded the flexural strength target of 25 MPa set by DOE. The tensile and flexural modulus of the composites increased with higher concentration of carbon fillers. Carbon black and synthetic graphite caused a decrease in the tensile and flexural strengths of the composites. However, carbon nanotubes increased the composite tensile and flexural strengths. Mathematical models were applied to estimate through-plane and in-plane thermal conductivities of single and multiple filler formulations, and tensile modulus of single-filler formulations. For thermal conductivity, Nielsen's model yielded accurate thermal conductivity values when compared to experimental results obtained through the Flash method. For prediction of tensile modulus Nielsen's model yielded the smallest error between the predicted and experimental values. The second part of this project consisted of the development of a curriculum in Fuel Cell and Hydrogen Technologies to address different educational barriers identified by the Department of Energy. By the creation of new courses and enterprise programs in the areas of fuel cells and the use of hydrogen as an energy carrier, we introduced engineering students to the new technologies, policies and challenges present with this alternative energy. Feedback provided by students participating in these courses and enterprise programs indicate positive acceptance of the different educational tools. Results obtained from a survey applied to students after participating in these courses showed an increase in the knowledge and awareness of energy fundamentals, which indicates the modules developed in this project are effective in introducing students to alternative energy sources.

Learning by the Dendritic Prediction of Somatic Spiking

Recent modeling of spike-timing-dependent plasticity indicates that plasticity involves as a third factor a local dendritic potential, besides pre- and postsynaptic firing times. We present a simple compartmental neuron model together with a non-Hebbian, biologically plausible learning rule for dendritic synapses where plasticity is modulated by these three factors. In functional terms, the rule seeks to minimize discrepancies between somatic firings and a local dendritic potential. Such prediction errors can arise in our model from stochastic fluctuations as well as from synaptic input, which directly targets the soma. Depending on the nature of this direct input, our plasticity rule subserves supervised or unsupervised learning. When a reward signal modulates the learning rate, reinforcement learning results. Hence a single plasticity rule supports diverse learning paradigms.

The role of post-translation regulation of Twist expression in the tumor progression of squamous cell carcinoma of head and neck

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://scansite.mit.edu/motifscan_seq.phtml ), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addition, mutation of the putative CK2 phosphorylation site (S18/S20A) in TWIST decreased the amount of phospho-ATP incorporated by TWIST in an in vitro kinase assay, and altered TWIST stability. In Boyd chamber migration assay and wound-healing assay, the CK2 inhibitor, DMAT, was found to decrease the motility of IL-6 stimulated SCCHN cells and over expression of either a wild-type or the hyperphosphorylated mimicking mutant S18/20D –Twist rather than the hypo-phosphorylated mimicking mutant S18/20A-Twist can promote SCCHN cell motility.To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

Tumor cell-associated epitopes recognized by lymphokine-activated killers (LAK)

Interleukin-2 activated lymphocytes, designated lymphokine-activated killers (LAK), acquire the unique capacity to express potent cytologic activity against a broad spectrum of abnormal and/or transformed NK-sensitive and NK-resistant target cells while sparing normal cell types. Investigations into the target spectra exhibited by cloned effector cells indicate that LAK cells express a polyspecific recognition mechanism that identifies an undefined class of cell surface-associated molecules expressed on susceptible targets. This report extends our previous investigations into the biochemical nature of these molecules by characterizing the functional role of two tumor cell-surface-associated epitopes implicated in conferring target cells with susceptibility to LAK-mediated cytotoxicity. The first moiety is implicated in the formation of effector/target cell conjugates. This binding ligand is preferentially expressed on tumor cells relative to LAK-resistant PBL target cells, sensitive to trypsin treatment, resistant to functional inactivation by heat- and detergent-induced conformational changes, and does not require N-linked glycosylation to maintain binding activity. In contrast, a carbohydrate-associated epitope represents the second tumor-associated molecule required for target cell susceptibility to LAK cells. Specifically, N-linked glyoprotein synthesis represents an absolute requirement for post-trypsin recovery of target cell susceptibility. The minimal N-linked oligosaccharide residue capable of restoring this second signal has been identified as a high mannose structure. Although ultimately required for tumor cell susceptibility, as measured in $\sp{51}$Cr-release assays, this N-glycan-associated molecule is not required for the differential tumor cell binding expressed by LAK cells. Furthermore, N-glycan expression is not adequate in itself to confer target cell susceptibility. Additional categories of cell surface components have been investigated, including O-linked oligosaccharides, and glycosaminoglycans, without identifying additional moieties relevant to target cell recognition. Collectively, these data suggest that tumor cell recognition by LAK cells is dependent on cell surface presentation of two epitopes: a trypsin-sensitive molecule that participates in the initial conjugate formation and an N-glycan-associated moiety that is involved in a post-binding event required for target cell killing. ^

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

CD8/CD45RO T-cell infiltration in endoscopic biopsies of colorectal cancer predicts nodal metastasis and survival

BACKGROUND AND AIMS: Reliable prognostic markers based on biopsy specimens of colorectal cancer (CRC) are currently missing. We hypothesize that assessment of T-cell infiltration in biopsies of CRC may predict patient survival and TNM-stage before surgery. METHODS: Pre-operative biopsies and matched resection specimens from 130 CRC patients treated from 2002-2011 were included in this study. Whole tissue sections of biopsy material and primary tumors were immunostained for pancytokeratin and CD8 or CD45RO. Stromal (s) and intraepithelial (i) T-cell infiltrates were analyzed for prediction of patient survival as well as clinical and pathological TNM-stage of the primary tumor. RESULTS: CD8 T-cell infiltration in the preoperative biopsy was significantly associated with favorable overall survival (CD8i p = 0.0026; CD8s p = 0.0053) in patients with primary CRC independently of TNM-stage and postoperative therapy (HR [CD8i] = 0.55 (95% CI: 0.36-0.82), p = 0.0038; HR [CD8s] = 0.72 (95% CI: 0.57-0.9), p = 0.0049). High numbers of CD8i in the biopsy predicted earlier pT-stage (p < 0.0001) as well as absence of nodal metastasis (p = 0.0015), tumor deposits (p = 0.0117), lymphatic (p = 0.008) and venous invasion (p = 0.0433) in the primary tumor. Infiltration by CD45ROs cells was independently associated with longer survival (HR = 0.76 (95% CI: 0.61-0.96), p = 0.0231) and predicted absence of venous invasion (p = 0.0025). CD8 counts were positively correlated between biopsies and the primary tumor (r = 0.42; p < 0.0001) and were reproducible between observers (ICC [CD8i] = 0.95, ICC [CD8s] = 0.75). For CD45RO, reproducibility was poor to moderate (ICC [CD45i] = 0.16, ICC [CD45s] = 0.49) and correlation with immune infiltration in the primary tumor was fair and non-significant (r[CD45s] = 0.16; p = 0.2864). For both markers, no significant relationship was observed with radiographic T-stage, N-stage or M-stage, indicating that assessment of T-cells in biopsy material can add additional information to clinical staging in the pre-operative setting. CONCLUSIONS: T-cell infiltration in pre-operative biopsy specimens of CRC is an independent favorable prognostic factor and strongly correlates with absence of nodal metastasis in the resection specimen. Quantification of CD8i is highly reproducible and allows superior prediction of clinicopathological features as compared to CD45RO. The assessment of CD8i infiltration in biopsies is recommended for prospective investigation.

Immunologic response to the survivin-derived multi-epitope vaccine EMD640744 in patients with advanced solid tumors

PURPOSE Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses. EXPERIMENTAL DESIGN This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide(®) ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 μg. Secondary objectives included safety, tolerability, and clinical efficacy. RESULTS In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event. CONCLUSIONS Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.