Outstanding negative prediction performance of solid pulmonary nodule volume AI for ultra-LDCT baseline lung cancer screening risk stratification.

OBJECTIVE: To evaluate performance of AI as a standalone reader in ultra-low-dose CT lung cancer baseline screening, and compare it to that of experienced radiologists. METHODS: 283 participants who underwent a baseline ultra-LDCT scan in Moscow Lung Cancer Screening, between February 2017-2018, and had at least one solid lung nodule, were included. Volumetric nodule measurements were performed by five experienced blinded radiologists, and independently assessed using an AI lung cancer screening prototype (AVIEW LCS, v1.0.34, Coreline Soft, Co. ltd, Seoul, Korea) to automatically detect, measure, and classify solid nodules. Discrepancies were stratified into two groups: positive-misclassification (PM); nodule classified by the reader as a NELSON-plus /EUPS-indeterminate/positive nodule, which at the reference consensus read was < 100 mm3, and negative-misclassification (NM); nodule classified as a NELSON-plus /EUPS-negative nodule, which at consensus read was ≥ 100 mm3. RESULTS: 1149 nodules with a solid-component were detected, of which 878 were classified as solid nodules. For the largest solid nodule per participant (n = 283); 61 [21.6 %; 53 PM, 8 NM] discrepancies were reported for AI as a standalone reader, compared to 43 [15.1 %; 22 PM, 21 NM], 36 [12.7 %; 25 PM, 11 NM], 29 [10.2 %; 25 PM, 4 NM], 28 [9.9 %; 6 PM, 22 NM], and 50 [17.7 %; 15 PM, 35 NM] discrepancies for readers 1, 2, 3, 4, and 5 respectively. CONCLUSION: Our results suggest that through the use of AI as an impartial reader in baseline lung cancer screening, negative-misclassification results could exceed that of four out of five experienced radiologists, and radiologists' workload could be drastically diminished by up to 86.7%.

Negative Predictive Value of Non-Echo-Planar Diffusion Weighted MR Imaging for the Detection of Residual Cholesteatoma Done at 9 Months After Primary Surgery Is not High Enough to Omit Second Look Surgery.

OBJECTIVES: To evaluate non echo-planar diffusion weighted magnetic resonance imaging (non-EP DW MRI) at 9 months after primary surgery to rule out residual cholesteatoma in patients scheduled before second-look-surgical exploration. STUDY DESIGN: Prospective observational study. SETTING: Secondary teaching hospital. PATIENTS/INTERVENTIONS: Patients who were scheduled for second-look-surgery after primary canal wall up repair of cholesteatoma underwent 1.5 T MRI including non-EP DWI and high-resolution coronal T1 and T2-FS SE sequences. MAIN OUTCOME MEASURES: Imaging studies were evaluated for the presence of cholesteatoma by three independent observers. Intraoperative observations were regarded the standard of reference. Ear, nose, throat (ENT) surgeons were blinded for imaging findings. The primary outcome was the negative predictive value (NPV) of MR imaging, secondary outcomes were sensitivity, specificity, and positive predictive value. RESULTS: Thirty-three patients underwent both MRI and surgery, among whom 22 had a cholesteatoma. Mean time between primary surgery and MRI was 259 days (standard deviation [SD] 108). NPV of non-EP DW MRI in detecting recurrent cholesteatoma was 53% (95% CI: 32-73%). Sensitivity and specificity were 59% (39-77%) and 91% (62-98%), respectively. The positive predictive value was 93% (69-99%). In five out of nine false-negative cases, recurrent cholesteatoma measured 3 mm or less. Using a 3 mm detection threshold, NPV increased to 79%. CONCLUSION: Non-EP DW MRI cannot replace second look surgery in ruling-out residual cholesteatoma at 9 months after primary surgery. It could be used in a follow-up strategy in low risk patients. Further research is needed which types of residual cholesteatoma are not revealed by MRI.

High-pitch versus sequential mode for coronary calcium in individuals with a high heart rate: Potential for dose reduction.

BACKGROUND: To determine the impact of high-pitch spiral acquisition on radiation dose and cardiovascular disease (CVD) risk stratification by coronary artery calcium (CAC) assessment with computed tomography in individuals with a high heart rate. METHODS: Of the ROBINSCA trial, 1990 participants with regular rhythm and heart rates >65 beats per minute (bpm) were included. As reference, 390 participants with regular heart rates ≤65 bpm were used. All participants underwent prospectively electrocardiographically(ECG)-triggered imaging of the coronary arteries using dual source CT at 120 kVp, 80 ref mAs using both high-pitch spiral mode and sequential mode. Radiation dose, Agatston score, number of positive scores, as well as median absolute difference of the Agatston score were determined and participants were stratified into CVD risk categories. RESULTS: A similar percentage of participants with low heart rates and high heart rates had a positive CAC score in data sets acquired in high-pitch spiral (low heart rate: 57.7%, high heart rate: 55.8%) and sequential mode (58.0%, 54.7%, p = n.s.). The median absolute difference in Agatston scores between acquisition modes was 14.2% and 9.2%, for the high and low heart rate groups, respectively. Excellent agreement for risk categorization between the two data acquisition modes was found for the high (κ = 0.927) and low (κ = 0.946) heart rate groups. Radiation dose was 48% lower for high-pitch spiral versus sequential acquisitions. CONCLUSION: Radiation dose for the quantification of coronary calcium can be reduced by 48% when using the high-pitch spiral acquisition mode compared to the sequential mode in participants with a regular high heart rate. CVD risk stratification agreement between the two modes of data acquisition is excellent.

CD4+ T-cell alloreactivity toward mismatched HLA class II alleles early after double umbilical cord blood transplantation.

Although double umbilical cord blood transplantation (dUCBT) in adult patients may be associated with less graft failure compared with single UCBT, hematopoietic recovery generally originates from a single cord blood unit (CBU). CBU predominance is still incompletely understood. We recently showed that blood CD4+ T-cell numbers rapidly increase after dUCBT, and early CD4+ T-cell chimerism predicts for graft predominance. Given the frequent HLA class II allele mismatches between CBUs in dUCBT, we hypothesized that alloreactive HLA class II-specific CD4+ T cells from the "winning" CBU may contribute to rejection of the "loser" CBU. We evaluated whether CD4+ T cells originating from the predominant (PD)-CBU would recognize HLA class II allele mismatches, expressed by the nonengrafting (NE)-CBU. Alloreactive effector CD4+ T cells toward 1 or more mismatched HLA class II alleles of the NE-CBU were detected in 11 of 11 patients, with reactivity toward 29 of 33 (88%) tested mismatches, and the strongest reactivity toward DR and DQ alleles early after dUCBT. Mismatched HLA class II allele-specific CD4+ T cells recognized primary leukemic cells when the mismatched HLA class II allele was shared between NE-CBU and patient. Our results suggest that cytotoxicity exerted by CD4+ T cells from the PD-CBU drives the rapid rejection of the NE-CBU, whose alloreactive effect might also contribute to graft-versus-leukemia.

[Referral for mammography often not according to guideline]. / Verwijzing voor mammogram vaak niet volgens richtlijn.

OBJECTIVE: To analyse the percentage of women with a family history of breast cancer referred by general practitioners (GPs) for a screening mammography in accordance with the Dutch Breast Cancer Guideline produced by the Netherlands Comprehensive Cancer Centre (IKNL). DESIGN: Prospective cohort study. METHOD: Women referred by their GP between December 2011 and December 2012 for mammography, with the indication "family history of breast cancer", were invited to take part in this study. A trained radiology laboratory assistant carried out a structured questionnaire to assess their risk on the basis of the categories of the 2008 IKNL guideline "Family history of breast/ovarian cancer". Based on the presence of certain risk factors, the women were allocated to one of the following groups: "referral for mammography", "referral to a clinical geneticist" or "no referral indicated". RESULTS: 242 women were referred by their GPs to the Radiology Department for mammography on the basis of family history; we included 210 women in our study. Their ages ranged from 25 to 77 years (mean age: 48 years). Forty-five patients (21%) were referred for mammography in accordance with the guideline. Twenty-two patients (10%) should have been referred to a clinical geneticist according to the guideline, whereas 143 patients (68%) did not meet the criteria for a screening mammography outside the screening programme. CONCLUSION: In only 21% of patients referred by their GPs for a screening mammography, with "family history" given as the reason, this referral was in accordance with the standard of the Dutch College of General Practitioners (NHG) or the IKNL guideline. Screening outside the breast cancer screening programme was not indicated according to the guideline for the majority of the women. Referral of 10% of the women referred should have been to a clinical geneticist; this figure rises to as many as 20% using the 2012 IKNL guideline.

Long-term stability of T-cell activation and transduction components critical to the processing of clinical batches of gene-engineered T cells.

BACKGROUND AIMS: The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan). METHODS: Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time. RESULTS: We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years. CONCLUSIONS: High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies.

Double umbilical cord blood transplantation: a study of early engraftment kinetics in leukocyte subsets using HLA-specific monoclonal antibodies.

Single cord blood unit (CBU) predominance is usually established within the first month after double umbilical cord blood transplantation (UCBT). However, the kinetics of engraftment of the different leukocyte subsets and the mechanism of graft predominance is largely unknown. To investigate whether a differential engraftment might reveal a specific subset that could play a key role in the mechanism of graft predominance, we studied early engraftment kinetics of different leukocyte subpopulations by flow cytometry using human monoclonal antigen-specific human leukocyte antigen antibodies, directed against mismatched human leukocyte antigen-A or -B antigens between recipient and CBUs. Twenty-two patients, who had received a double UCBT preceded by a reduced-intensity conditioning regimen, were evaluated at days +11, +18, +25, and +32 posttransplantation. Single CBU predominance in the various leukocyte subsets was established within 18 days posttransplantation. CD4+ T cells of the dominant CBU showed early peripheral blood expansion. Moreover, chimerism in CD4+ and CD8+ T cell and natural killer cell subsets at day +11 was predictive of ultimate graft predominance. These findings show that engraftment kinetics of the various leukocyte subsets vary considerably after double UCBT and may suggest an important role for CD4+ T cells in a presumed alloreactive graft-versus-graft rejection.

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue.

Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic™), real-time PCR (EntroGen™) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.

Inflammation and fatigue dimensions in advanced cancer patients and cancer survivors: an explorative study.

BACKGROUND: Inflammation may underlie cancer-related fatigue; however, there are no studies that assess the relation between fatigue and cytokines in patients with advanced disease versus patients without disease activity. Furthermore, the relation between cytokines and the separate dimensions of fatigue is unknown. Here, association of plasma levels of inflammatory markers with physical fatigue and mental fatigue was explored in advanced cancer patients and cancer survivors. METHODS: A total of 45 advanced cancer patients and 47 cancer survivors completed the subscales Physical Fatigue and Mental Fatigue of the Multidimensional Fatigue Inventory. Plasma concentrations of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1-ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and neopterin were measured. Nonparametric tests were used to assess differences in fatigue intensity and levels of inflammatory markers and to determine correlation coefficients between the fatigue dimensions and inflammatory markers. RESULTS: Compared with cancer survivors, patients with advanced cancer had higher levels of physical fatigue (median 16 vs 9, P < .001) and mental fatigue (median 11 vs 6, P = .01). They also had higher levels of all cytokines (P < .01). In advanced cancer, CRP (r = 0.49, P = .001), IL-6 (r = 0.43, P = .003), IL-1-ra (r = 0.32, P = .03), and neopterin (r = 0.25, P = .10) were correlated with physical but not with mental fatigue. In cancer survivors, only IL-1-ra was related to both physical fatigue (r = 0.24, P = .10) and mental fatigue (r = 0.35, P = .02). CONCLUSIONS: In advanced cancer, inflammation seems to be associated with physical fatigue, but not to mental fatigue. In cancer survivors, there was no convincing evidence that inflammation plays a major role in fatigue.

Insufficient recovery of thymopoiesis predicts for opportunistic infections in allogeneic hematopoietic stem cell transplant recipients.

BACKGROUND: Recovery of thymopoiesis after allogeneic hematopoietic stem cell transplantation is considered pivotal for full immune competence. However, it is still unclear to what extent insufficient recovery of thymopoiesis predicts for subsequent opportunistic infections and non-relapse mortality. DESIGN AND METHODS: A detailed survey of all post-engraftment infectious complications, non-relapse mortality and overall survival during long-term follow-up was performed in 83 recipients of allogeneic stem cell grafts after myeloablative conditioning. Recovery of thymopoiesis was assessed using analysis of signal joint T-cell receptor rearrangement excision circles. The impact of recovery of thymopoiesis at 2, 6, 9 and 12 months post-transplantation on clinical outcome beyond those time points was evaluated by univariate and multivariate Cox regression analyses. RESULTS: The cumulative incidence of severe infections at 12 months after transplantation was 66% with a median number of 1.64 severe infectious episodes per patient. Patients in whom thymopoiesis did not recover were at significantly higher risk of severe infections according to multivariable analysis. Hazard ratios indicated 3- and 9-fold increases in severe infections at 6 and 12 months, respectively. Impaired recovery of thymopoiesis also translated into a higher risk of non-relapse mortality and outweighed pre-transplant risk factors including age, donor type, and disease risk-status. CONCLUSIONS: These results indicate that patients who fail to recover thymopoiesis after allogeneic hematopoietic stem cell transplantation are at very high risk of severe infections and adverse clinical outcome.

Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146.

Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normal-like" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61-66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.

mRNA levels of CD31, CD144, CD146 and von Willebrand factor do not serve as surrogate markers for circulating endothelial cells.

Circulating endothelial cells (CEC) are considered a promising marker to determine the extent of vascular damage. However, currently available and validated CEC enumeration assays are laborious, time consuming and costly, which limits their clinical utility. Here, we evaluated the feasibility of quantifying mRNA levels of the endothelium-associated markers CD31, CD144, CD146 and von Willebrand factor (vWf) in peripheral blood (PB) of healthy donors, patients, and human umbilical veins by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and their use as surrogate markers for CEC. Whole blood samples and CD146+ cell-enriched fractions were assessed for mRNA and protein expression of CD31, CD144, CD146 and vWf by RT-PCR and flow cytometry, respectively. We showed the feasibility to detect endothelial mRNA isolated from HUVEC numbers as low as 10. However, no endothelial mRNA could be measure in whole blood samples, and only low levels of CD31 and CD146 mRNA were detected in suspensions of isolated CEC with numbers up to 4,450 CEC per sample. We conclude that mRNA levels of CD31, CD144, CD146 and vWf in whole blood as detected by real time RT-PCR cannot be used as biomarkers for end-stage endothelial cells such as CEC.

Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer.

The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool, as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies. However, implementation of this assay into clinical routine has been cumbersome, as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers, and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step. This review addresses the assays that have been described so far in the literature, including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients.

Gibbon ape leukemia virus poorly replicates in primary human T lymphocytes: implications for safety testing of primary human T lymphocytes transduced with GALV-pseudotyped vectors.

The Food and Drug Administration/Center for Biologics Evaluation and Research has defined that for retroviral gene therapy, the vector-producing cell, the vector preparation, and the ex vivo gene-transduced cells have to be tested for absence of replication-competent retrovirus (RCR) if the transduced cells are cultured for >4 days. We assessed the sensitivity of the "extended PG4(S+L-) assay" to detect gibbon ape leukemia virus (GALV) RCR, and applied this assay to measure GALV RCR spread in retrovirally transduced T cells. To this end, T cells were expanded for 12 days after transduction with a GALV-envelope pseudotyped retroviral vector expressing single chain variable fragment (anticarbonic anhydrase IX) in presence or absence of GALV RCR. Results showed that: (1) the "extended PG4(S+L-) assay" detects 1 focus-forming unit (ffu) GALV RCR and thus is applicable and sufficiently sensitive to screen human T-cell cultures for absence of infectious GALV RCR; (2) although GALV RCR infect human T cells, it very poorly replicate in T cells; (3) GALV RCR, when present at low levels immediately upon gene transduction (ie, 100 ffu/20x10 T cells in 100 mL), did not spread during a 12-day T-cell culture at clinical scale. Our observation that GALV RCR poorly spreads in primary human T-cell cultures questions the relevance of testing T-cell transductants for RCR on top of testing the vector-producing cells and the clinical vector batch for RCR and warrants evaluation of the current policy for safety testing of ex vivo retrovirally transduced T lymphocytes for GALV RCR.

Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells.

Identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all tumor cell types. We investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling-normal-like, basal, HER2-positive, and luminal A and B-were identified by CellSearch, a US Food and Drug Administration-approved test that uses antibodies against the cell surface-expressed epithelial cell adhesion molecule (EpCAM) to isolate circulating tumor cells. We used global gene expression profiling to determine the subtypes of a well-defined panel of 34 human breast cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single healthy human donor, and the mixtures were subjected to the CellSearch test to isolate the breast cancer cells. We found that the CellSearch isolation method, which uses EpCAM on the surface of circulating tumor cells for cell isolation, did not recognize, in particular, normal-like breast cancer cells, which in general have aggressive features. New tests that include antibodies that specifically recognize normal-like breast tumor cells but not cells of hematopoietic origin are needed.

Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR.

Detection of circulating tumor cells (CTCs) in whole blood from metastatic cancer patients by the CellSearch CTC Test (Veridex LLC, Warren, NJ, USA) has been shown to have clinical relevance. In addition to enumeration, there is great interest in molecular characterization of these CTCs. We aimed to establish a robust method to perform mRNA expression analysis of multiple genes by a real-time reverse transcriptase (RT)-PCR on small numbers of CTCs enriched from whole blood by the CellSearch system. Despite the 4 log depletion of leukocytes after CellSearch enrichment, the CTC-enriched fractions still contained leukocytes, in particular B-lymphocytes, which severely interfered with our CTC-specific gene expression profiling. After extensive washing and leukocyte-specific depletion by anti-CD45 coated magnetic beads prior to CellSearch enrichment, the number of leukocytes present in the enriched fraction was still high (range 60-929). However, by using a set of genes with no or minor expression by leukocytes, we succeeded to perform quantitative gene expression profiling specific for as little as one breast cancer CTC present in a CTC-enriched environment typically containing over 800 contaminating leukocytes. Our method allows molecular characterization specific for as little as one CTC, and can be used to expand the understanding of the biology of metastasis and, potentially, to improve patient management.

Selection of human antibody fragments directed against tumor T-cell epitopes for adoptive T-cell therapy.

Adoptive transfer of antigen-specific T-cells has shown therapeutic successes in the treatment of tumors in patients with metastatic melanoma. Tumor antigen-specific T-lymphocytes, however, occur only at low frequencies in a small proportion of patients. This low T-lymphocyte frequency together with the difficulties associated with in vitro generation of T-lymphocytes specific for cancers other than melanoma hampers adoptive T cell therapy. To make adoptive T-cell therapy more uniformly applicable, strategies were developed at transferring tumor-specificity to primary human T-lymphocytes via antibody (Ig) or T-cell receptor (TCR) molecules. We exploited the selection power of phage display that allows for the testing of tens of billions of individual clones with a high-throughput selection of Fabs with peptide/MHC complex binding capacity. Following in vitro selection, human "TCR-like" Fab fragments have been functionally expressed on human T-lymphocytes, resulting in MHC-restricted, tumor-specific lysis and cytokine production. Currently, we have extended our selections to a panel of class I and II MHC-restricted MAGE and other tumor-specific epitopes, and would like to propose that phage display represents a technology able to expand T-cell therapy to numerous tumor types.

B and T cell imbalances in CSF of patients with Hu-antibody associated PNS.

In paraneoplastic neurological syndromes associated with Hu-antibodies (Hu-PNS) an important role for cellular immunity is hypothesized. We characterized the cerebrospinal fluid (CSF) pleocytosis in Hu-PNS patients by assessing the major lymphocyte subsets by flow cytometry. The B cell subset in the CSF of Hu-PNS patients showed a significant absolute (approximately 20x) and relative (approximately 3x) expansion, while the numbers of CD4+ T cells, CD8+ T cells and NK cells only showed an absolute expansion (approximately 4-7x) compared to the controls. On the other hand, the NKT cell subset showed a significant relative reduction in CSF and in blood of Hu-PNS patients. The relative B cell expansion is consistent with the intrathecal synthesis of Hu-antibodies, while the increased number of T and NK cells supports an additional role for cellular immunity in the pathogenesis of Hu-PNS. In addition, the autoimmune hypothesis of Hu-PNS is supported by the relative NKT cell deficiency.

Circulating tumour cell detection on its way to routine diagnostic implementation?

Circulating tumour cells (CTCs) have been considered for a long time in reflecting the aggressiveness of tumours. As a result, many attempts have been made to develop assays that reliably detect and enumerate CTCs, but only recently have such assays been available. The first clinical results obtained with such assays strongly suggest that in some tumour types, CTC detection and enumeration can be used to estimate prognosis and may serve as an early marker to assess anti-tumour activity of a treatment. Furthermore, through technical advances, CTCs can be characterised for several features, which may shortly yield better prognostic and predictive classification systems and may also provide improved insight into biological processes including dissemination, drug resistance and treatment-induced cell death. This review addresses CTCs, and in particular, technical issues concerning their detection, clinical results obtained so far, and future perspectives.

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo.

BACKGROUND: We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)(+) T cells is reflected by changes of selected immune parameters measured in peripheral blood. METHODS: ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)(+) T lymphocytes prior to infusion, and were correlated with plasma cytokine levels. RESULTS: Prior to infusion, scFv(G250)(+) T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX(+) RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-gamma were produced, whilst production of TNF-alpha, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-gamma was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-gamma increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2. CONCLUSION: ScFv(G250)-receptor-mediated functions of the scFv(G250)(+) T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-gamma levels.