Addition of FFRct in the diagnostic pathway of patients with stable chest pain to reduce unnecessary invasive coronary angiography (FUSION) : Rationale and design for the multicentre, randomised, controlled FUSION trial.

BACKGROUND: Coronary computed tomography angiography (CCTA) is widely used in the diagnostic work-up of patients with stable chest pain. CCTA has an excellent negative predictive value, but a moderate positive predictive value for detecting coronary stenosis. Computed tomography-derived fractional flow reserve (FFRct) is a non-invasive, well-validated technique that provides functional assessment of coronary stenosis, improving the positive predictive value of CCTA. However, to determine the value of FFRct in routine clinical practice, a pragmatic randomised, controlled trial (RCT) is required. We will conduct an RCT to investigate the impact of adding FFRct analysis in the diagnostic pathway of patients with a coronary stenosis on CCTA on the rate of unnecessary invasive coronary angiography, cost-effectiveness, quality of life and clinical outcome. METHODS: The FUSION trial is a prospective, multicentre RCT that will randomise 528 patients with stable chest pain and anatomical stenosis of ≥ 50% but < 90% in at least one coronary artery of ≥ 2 mm on CCTA, to FFRct-guided care or usual care in a 1:1 ratio. Follow-up will be 1 year. The primary endpoint is the rate of unnecessary invasive coronary angiography within 90 days. CONCLUSION: The FUSION trial will evaluate the use of FFRct in stable chest pain patients from the Dutch perspective. The trial is funded by the Dutch National Health Care Institute as part of the research programme 'Potentially Promising Care' and the results will be used to assess if FFRct reimbursement should be included in the standard health care package.

Ultrasound-guided breast-conserving surgery for early-stage palpable and nonpalpable invasive breast cancer: decreased excision volume at unchanged tumor-free resection margin.

Ultrasound guidance (USG) during breast-conserving surgery improves tumor-free surgical resection margins. The objective of this study was to evaluate whether USG reduces resection volumes without compromising margin status. 134 patients with palpable or nonpalpable T1-2N0-1 invasive breast cancer were treated with USG and compared with a historical reference control group (CON) consisting of palpation-guided (PAG) or wire-guided localization (WIG) breast-conserving surgery. Primary outcomes were excess resection volume and clear margin status, and secondary outcome was re-excision rate. 66 patients underwent USG. In the CON group (n = 68), PAG was performed in 24 (35 %) and WIG in 44 (64 %) patients. Median excision volume [39 (IQR 20-66) vs 56 (38-94) cm(3); p = 0.001] and median calculated resection ratio [1.7 (1.0-2.9) vs 2.8 (1.4-4.6) (p = 0.005)] were significantly smaller in the USG than in the CON group. Median minimal distance to the resection margin [4 mm (IQR 2-5 mm) vs 2 mm (1-4 mm), p = 0.004] was significantly larger. Clear resection margins were achieved in 58 of the USG patients (88 %) and in 58 of the CON patients (86 %) (p = 0.91); this was true in patients with palpable as well as nonpalpable lesions. Reexcision was needed in 6.1 and 7.2 % respectively. Relative risk for re-excision in the USG group was 0.82 (95 % CI 0.23-2.93). In patients with palpable and nonpalpable breast cancers, USG allows for lower excision volume and reduced resection of healthy breast tissue, without increased re-excision rate.

Prognostic value of circulating tumour cells for early recurrence after resection of colorectal liver metastases.

BACKGROUND: Despite good outcomes for many, a substantial group of patients undergoing metastasectomy for isolated liver metastases from colorectal cancer (CRC) experience early recurrence. We have investigated whether circulating tumour cell (CTC) detection can identify patients developing disease recurrence within 1 year after liver metastasectomy. METHODS: In CRC patients undergoing liver metastasectomy, 30 ml peripheral blood was withdrawn preoperatively. CTCs were detected by the CellSearch system after a density-gradient-based enrichment step. RESULTS: One hundred and seventy-three samples from 151 individual patients were analysed. In 75 samples (43%), CTCs were detected, 16% had ⩾3 CTCs/7.5 ml of blood. Eighty-two patients (47%) experienced early disease recurrence (<1 year). The 1-year recurrence rate between patients with or without detectable CTCs were similar (47% vs 48%) or with a low or high CTC count (<3 or ⩾3 CTCs/7.5 ml of blood) (50% vs 47%). Also disease-free and overall survival were similar between patients with or without CTCs. CONCLUSIONS: The presence of CTCs in preoperative peripheral blood samples does not identify patients at risk for early disease recurrence after curative resection of colorectal liver metastases. Other parameters are needed to better identify patients at high risk to relapse after liver metastasectomy for CRC.

Prognostic value and kinetics of circulating endothelial cells in patients with recurrent glioblastoma randomised to bevacizumab plus lomustine, bevacizumab single agent or lomustine single agent. A report from the Dutch Neuro-Oncology Group BELOB trial.

BACKGROUND: Angiogenesis is crucial for glioblastoma growth, and anti-vascular endothelial growth factor agents are widely used in recurrent glioblastoma patients. The number of circulating endothelial cells (CECs) is a surrogate marker for endothelial damage. We assessed their kinetics and explored their prognostic value in patients with recurrent glioblastoma. METHODS: In this side study of the BELOB trial, 141 patients with recurrent glioblastoma were randomised to receive single-agent bevacizumab or lomustine, or bevacizumab plus lomustine. Before treatment, after 4 weeks and after 6 weeks of treatment, CECs were enumerated. RESULTS: The number of CECs increased during treatment with bevacizumab plus lomustine, but not during treatment in the single-agent arms. In patients treated with lomustine single agent, higher absolute CEC numbers after 4 weeks (log10CEC hazard ratio (HR) 0.41, 95% CI 0.18-0.91) and 6 weeks (log10CEC HR 0.16, 95% CI 0.05-0.56) of treatment were associated with improved overall survival (OS). Absolute CEC numbers in patients receiving bevacizumab plus lomustine or bevacizumab single agent were not associated with OS. CONCLUSION: CEC numbers increased during treatment with bevacizumab plus lomustine but not during treatment with either agent alone, suggesting that this combination induced the greatest vascular damage. Although the absolute number of CECs was not associated with OS in patients treated with bevacizumab either alone or in combination, they could serve as a marker in glioblastoma patients receiving lomustine single agent.

Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients.

BACKGROUND: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS: CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION: A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

Endothelial CD276 (B7-H3) expression is increased in human malignancies and distinguishes between normal and tumour-derived circulating endothelial cells.

BACKGROUND: Mature circulating endothelial cells (CEC) are surrogate markers of endothelial damage. CEC measured in patients with advanced cancer are thought not only to derive from damaged normal vasculature (n-CEC), but also from damaged (t-CEC). Therefore, assays that allow the discrimination between these two putative types of CEC are thought to improve the specificity of the enumeration of CEC in cancer. METHODS: Identification of tumour-associated endothelial markers (TEM) by comparing antigen expression on normal vs t-CEC and assess the presence of t-CEC in peripheral blood of cancer patients by incorporating TEM in our novel flow cytometry-based CEC detection assay. RESULTS: No difference in antigen expression between normal and malignant endothelial cells (ECs) was found for CD54, CD109, CD137, CD141, CD144 and CXCR7. In contrast, overexpression for CD105, CD146, CD276 and CD309 was observed in tumour ECs compared with normal ECs. CD276 was most differentially expressed and chosen as a marker for further investigation. CD276-expressing CEC were significantly higher in 15 patients with advanced colorectal cancer (median 9 (range 1-293 cell per 4 ml); P<0.005), in 83 patients with a glioblastoma multiforme (median 10 (range 0-804); P<0.0001) and in 14 patients with advanced breast cancer (median 14 (range 0-390) P<0.05) as compared with 24 healthy individuals (median 3 (range 0-11)). Of all patients with malignancies, 58% had CD276(+) CEC counts above the ULN (8 cell per 4 ml). CONCLUSIONS: The present study shows that CD276 can be used to discriminate ECs from malignant tissue from ECs from normal tissue. In addition, CD276(+) CEC do occur in higher frequencies in patients with advanced cancer.

High prevalence of abdominal aortic aneurysm in older men with cerebrovascular disease: Evaluation of a local screening program.

Introduction: Patients with cerebrovascular disease may suffer from other vascular morbidities, such as abdominal aortic aneurysm (AAA). Previously, a high prevalence of AAA has been demonstrated in men 60 years of age and older who have experienced TIA or stroke. This report evaluates the results of a decade's operation of a local screening program for AAA in this selected neurologic population. Methods: Men aged ≥60 years and admitted to the neurology ward of a community-based hospital in the Netherlands from 2006 to 2017 with a diagnosis of TIA or stroke were selected for screening. The diameter of the abdominal aorta was assessed by abdominal ultrasonography. Patients with detected AAA were referred for evaluation by a vascular surgeon. Results: AAA was detected in 72 of 1,035 screened patients (6.9%). AAAs with a diameter of 3.0-3.9 cm accounted for 61.1% of the total aneurysms found; AAAs with a diameter of 4.0-5.4 cm accounted for 20.8% of the total; and large aneurysms with a diameter of ≥5.5 cm accounted for 18.1% of all aneurysms discovered. A total of 18 patients (1.7%) underwent elective aneurysm repair. Discussion: The detection rate of AAA in older men with cerebrovascular disease was roughly 5-fold the detection rate in known European screening programs in older men from the general population. The proportion of large AAAs (≥5.5 cm) was also substantially higher. These findings reveal a previously unknown co-morbidity in patients with cerebrovascular disease and may be helpful for cardiovascular management of this large group of neurologic patients. Current and future AAA screening programs may also benefit from this knowledge.

Human chorionic gonadotropin treatment of anti-Hu-associated paraneoplastic neurological syndromes.

OBJECTIVE: Paraneoplastic neurological syndromes associated with anti-Hu antibodies (Hu-PNS) are mediated by a T-cell immune response that is directed against the Hu antigens. In pregnancy, many Th1-mediated autoimmune diseases such as rheumatoid arthritis and multiple sclerosis regress. We hypothesised that this decreased disease activity during pregnancy may be related to high human chorionic gonadotropin (hCG) levels. METHODS: 15 Hu-PNS patients were treated in a prospective, uncontrolled and unblinded trial with 10,000 IU daily of hCG administered by intramuscular injection during 12 weeks. Primary outcome measures were functional improvement defined as a decrease of one or more points on the modified Rankin Scale (mRS) or stabilisation in patients with mRS score ≤3 and improvement of neurological impairment assessed with the Edinburgh Functional Impairment Tests (EFIT). Secondary end points included the change in activities of daily living as evaluated using the Barthel Index. RESULTS: Seven of 15 patients (47%) improved on the mRS or stabilised at mRS score ≤3. Four patients (27%) showed significant improvement of neurological impairment as indicated by an overall Edinburgh Functional Impairment Tests score of ≥1 point. Five patients improved on the Barthel Index (33%). CONCLUSION: Comparison with previous studies suggests that hCG may have immunomodulatory activity and may modify the course of Hu-PNS, although well-established confounding factors may have contributed in this uncontrolled trial.

Screening for aneurysm of the abdominal aorta: prevalence in patients with stroke or TIA.

BACKGROUND AND PURPOSE: Population screening for aneurysms of the abdominal aorta (AAA) is still not implemented in any country, despite proven benefit both in decreased mortality and in cost effectiveness. Detecting a subpopulation with higher prevalence of AAA may alter this situation. METHODS: Between 2002 and 2005, all patients with a stroke or transient ischaemic attack (TIA) admitted to the department of Neurology of a community-based hospital were classified according to the Toast criteria and enrolled in a prospective study to assess the diameter of the abdominal aorta. The diameter was assessed by ultrasonography. A written questionnaire and blood tests were used to assess risk factors. RESULTS: The prevalence of AAA amongst the 499 screened patients in the study was 5.8% [95% confidence interval (CI) 5.6-6.0%]. Of the risk factors or Toast criteria, only male gender and age over 59 years correlated significantly with AAA. In the subgroup of 235 men aged over 59 years, the prevalence of AAA was 11.1% (95% CI 10.4-11.8%). CONCLUSION: The prevalence of AAA in men over 59 years of age presenting with a stroke or TIA is nearly twofold increased (11.1%) compared with all patients. Therefore, screening for AAA in this subgroup of patients seems beneficial. However, further studies are needed to confirm this finding and to explore the clinical benefit and cost effectiveness.

Circulating endothelial cells in oncology: pitfalls and promises.

Adequate blood supply is a prerequisite in the pathogenesis of solid malignancies. As a result, depriving a tumour from its oxygen and nutrients, either by preventing the formation of new vessels, or by disrupting vessels already present in the tumour, appears to be an effective treatment modality in oncology. Given the mechanism by which these agents exert their anti-tumour activity together with the crucial role of tumour vasculature in the pathogenesis of tumours, there is a great need for markers properly reflecting its impact. Circulating endothelial cells (CEC), which are thought to derive from damaged vasculature, may be such a marker. Appropriate enumeration of these cells appears to be a technical challenge. Nevertheless, first studies using validated CEC assays have shown that CEC numbers in patients with advanced malignancies are elevated compared to healthy controls making CEC a potential tool for among other establishing prognosis and therapy-induced effects. In this review, we will address the possible clinical applications of CEC detection in oncology, as well as the pitfalls encountered in this process.

Retronectin-assisted retroviral transduction of primary human T lymphocytes under good manufacturing practice conditions: tissue culture bag critically determines cell yield.

BACKGROUND: For our clinical immunogene therapy study for the treatment of renal cell carcinoma (RCC) patients, we had developed a protocol for gene transduction and expansion of human T cells in compliance with good manufacturing practice (GMP) criteria. Critical to our successful clinical-scale transductions of patient T cells was the use of Retronectin in combination with Lifecell X-foldtrade mark cell culture bags. METHODS: In our current study, we evaluated two alternative types of bags for the Retronectin-mediated retroviral transduction of human T cells: the Miltenyi DC-generation bag and the Takara CultiLife Spin bag. RESULTS: In static transductions, but not in spinoculation, the DC-generation bags and CultiLife Spin bags performed as well as Lifecell X-foldtrade mark bags in Retronectin-assisted retroviral transduction of human T cells with respect to transduction efficiency, lymphocyte subset composition and lymphocyte function. However, both types of bags performed less well than Lifecell X-foldtrade mark cell culture bags in terms of cell yield. DISCUSSION: Adjusted numbers of cells at the start of transduction should be used when using the Miltenyi or Takara bags in order to compensate for the lower cell yield following transduction.

Bone-marrow transplantation fails to halt intrathecal lymphocyte activation in multiple sclerosis.

BACKGROUND: Given the presumed key role for autoreactive lymphocytes in multiple sclerosis (MS), treatment strategies have been developed to ablate lymphocyte activity. Intrathecal lymphocyte activation can be measured by CSF-soluble(s)CD27. OBJECTIVE: To determine the effect of maximum whole-body immune ablation on two different markers that detect lymphocyte activation in CSF-oligoclonal IgG bands and levels of CSF-sCD27. DESIGN, SETTING AND PATIENTS: The study quantified sCD27 levels and assessed the presence of oligoclonal IgG bands in CSF samples of secondary progressive patients with MS treated by autologous bone-marrow transplantation. In eight individuals, CSF was taken before and 6-9 months after conditioning. CSF-sCD27 levels were compared with other MS and non-inflammatory neurological disease controls. Regarding the effect of stem-cell transplantation on CSF oligoclonal bands, the study analysed pooled data of this and four other international studies on stem-cell transplantation in MS. RESULTS: CSF-sCD27 was significantly lower after the extremely immunoablative protocol. However, levels remained elevated compared with non-inflammatory controls and stayed within the range observed in other MS controls. The joint analysis of CSF oligoclonal bands demonstrated persistence of this immune abnormality in 88% of the reported cases (n = 34). CONCLUSIONS: The persistence of CSF lymphocyte activation markers sCD27 and intrathecal oligoclonal IgG bands after maximum immunoablative treatment indicates that complete eradication of activated lymphocytes from the CNS has not been established. This is paralleled by disease progression observed in several studies on the effect of stem-cell transplantation in MS.

Discriminant function analysis as decision support system for the diagnosis of acute leukemia with a minimal four color screening panel and multiparameter flow cytometry immunophenotyping.

Despite several recommendations for standardization of multiparameter flow cytometry (MFC) the number, specificity and combinations of reagents used by diagnostic laboratories for the diagnosis and classification of acute leukemias (AL) are still very diverse. Furthermore, the current diagnostic interpretation of flow cytometry readouts is influenced arbitrarily by individual experience and knowledge. We determined the potential value of a minimal four-color combination panel of 13 monoclonal antibodies (mAbs) with a CD45/sideward light scatter-gating strategy for a standardized MFC immunophenotyping of the clinically most relevant subgroups of AL. Bone marrow samples from 155 patients with acute myeloid leukemia (AML, n=79), B-cell precursor acute lymphoblastic leukemia (BCP-ALL, n=29), T-cell precursor acute lymphoblastic leukemia (T-ALL, n=12) and normal bone marrow donors (NBMD, n=35) were analyzed. A knowledge-based learning algorithm was generated by comparing the results of the minimal panel with the actual diagnosis, using discriminative function analysis. Correct classification of the test sample according to lineage, that is, BCP-ALL, T-ALL, AML and differentiation of NBMD was achieved in 97.2% of all cases with only six of the originally applied 13 mAbs of the panel. This provides evidence that discriminant function analysis can be utilized as a decision support system for interpretation of flow cytometry readouts.

[Pneumatosis intestinalis; no disease, but a symptom]. / Pneumatosis intestinalis; geen ziekte, maar een symptoom.

In three patients, men aged 77, 83 and 69 years, pneumatosis intestinalis was detected during CT for abdominal pain occurring in the first patient after an aortic stent had been placed, and during laparotomy because of ileus in the latter two patients. The first patient underwent removal of an ischaemic intestinal segment but died later due to infection around the prosthesis. The other two patients recovered after conservative therapy. Pneumatosis intestinalis is defined as the presence of gas in the wall of the gastrointestinal tract. Often it is detected by accident during abdominal radiographic examination or laparotomy. Pneumatosis intestinalis is a symptom and has been found in a wide variety of diseases. The clinical condition of the patient and the underlying disease determine the clinical significance of pneumatosis intestinalis and the therapy. The main issue is whether surgical intervention is necessary because of intestinal ischaemia or perforation.

Blood volume and body fluid compartments in lambs with aortopulmonary left-to-right shunts.

A left-to-right shunt is accompanied by an increased plasma and blood volume. Since this is likely realized through renin/aldosterone-mediated salt and water retention, other body fluid compartments may be changed too. Therefore, we studied blood volume and body fluid compartments by a single-injection, triple-indicator dilution technique in nine 8-wk-old lambs with an aortopulmonary left-to-right shunt (55 +/- 3% of left ventricular output; mean +/- SEM) and in 11 control lambs, 2.5 wk after surgery. Systemic blood flow was maintained at the same level as in control lambs, but the aortic pressure of the shunt lambs was lower. Blood volume in shunt lambs was larger than in control lambs (110 +/- 6 vs. 84 +/- 7 ml/kg, P < 0.001) through an increase in plasma volume, which correlated significantly with the magnitude of the left-to-right shunt (r = 0.81, P < 0.01). Red blood cell volume was equal to that of control lambs. Evidence was obtained that the increase in plasma volume was induced by a transient increase in renin (8.0 +/- 2.2 vs. 1.6 +/- 0.2 nmol.l-1.h-1; P < 0.02) and aldosterone (0.51 +/- 0.14 vs. 0.24 +/- 0.09 nmol/liter) concentrations. Interstitial water volume, however, was not significantly different from that in control lambs. The amount of intravascular protein was significantly higher than in control lambs (5.0 +/- 0.3 vs. 3.5 +/- 0.2 g/kg body mass, P < 0.001). There were no significant differences in intracellular and total body water volumes between the two groups. We conclude that the increased amount of intravascular protein confines the fluid retained by the kidneys to the vascular compartment.

Imbalances in circulating lymphocyte subsets in Hu antibody associated paraneoplastic neurological syndromes.

In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies, neuron-specific Hu antigens expressed by the tumour hypothetically trigger an immune response that cross-reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized cell-mediated immune pathogenesis of these syndromes, we analysed the circulating lymphocyte subsets in untreated patients with SCLC, PNS and Hu antibodies (n = 18), SCLC without PNS (n = 19) and controls (n = 29) using flow cytometry. SCLC patients with PNS had a variety of imbalances within their circulating lymphocyte subsets as compared with SCLC patients without PNS and healthy controls: (i) a lymphopenia of the major subsets (i.e. B, CD4+ and CD8+ T lymphocytes); (ii) increased proportions of activated CD4+ and CD8+ T cells; (iii) reduced numbers of terminally differentiated effector CD8+ T cells and cells with a cytotoxic T-cell phenotype (CD56+ and CD57+). Although indirect, our data provide further support for the involvement of T cells in the pathogenesis of Hu antibody associated PNS.

Systematic review of the safety and efficacy of contrast injection via venous catheters for contrast-enhanced computed tomography.

OBJECTIVE: To examine the safety and efficacy of contrast injection through a central venous catheter (CVC) for contrast-enhanced computed tomography (CECT). METHODS: A systematic literature search was performed using PubMed. Studies were deemed eligible if they reported on the use of CVCs for contrast administration. Selected articles were assessed for their relevance and risk of bias. Articles with low relevance and high risk of bias or both were excluded. Data from included articles was extracted. RESULTS: Seven studies reported on the use of CVCs for contrast administration. Catheter rupture did not occur in any study. The incidence of dislocation ranged from 2.2-15.4%. Quality of scans was described in three studies, with less contrast enhancement of pulmonary arteries and the thoracic aorta in two studies, and average or above average quality in one study. Four other studies used higher flowrates, but did not report quality of scans. CONCLUSION: Contrast injection via CVCs can be performed safely for CECT when using a strict protocol. Quality of scans depended on multiple factors like flow rate, indication of the scan, and cardiac output of the patient. In each patient, an individual evaluation whether to use the CVC as access for contrast media should be made, while bolus tracking may be mandatory in most cases.

Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer.

We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30-60% scFv(G250)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10-30% higher levels of scFv(G250)-mediated TNFalpha production and cytolysis of G250L+ RCC cells than T cells transduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

T-lymphocyte reconstitution following rigorously T-cell-depleted versus unmodified autologous stem cell transplants.

We compared the kinetics of T-cell recovery after extensive ex vivo and in vivo T-cell depleted autologous stem cell transplantation (SCT) for multiple sclerosis (MS; n=8) with unmodified SCT for hematological malignancies (HM; n=39). Both patient group showed a very protracted recovery of 'naive' CD4(+), 45R0(-) ( approximately CD45RA(+)) T-cells. Within the 'primed' CD4(+), 45R0(+) T-cells, the 'central memory' cells expressing the CD62L and CD27 markers were the slowest to recover. The repopulating T-cells were highly activated, as shown by increased expression of HLA-DR and the apoptosis marker CD95. The capability of CD4(+) and CD8(+) T-cells to produce IFN-gamma, IL-2 and TNF-alpha had reached normal ranges from 2 months post SCT onwards. Unexpectedly, the kinetics of T-cell recovery between 3 and 12 months post transplant was similar in T-depleted and unmodified SCT. Before SCT, the HM patients showed lymphopenia of all T-cell subsets, upregulated HLA-DR and CD95 expression and increased cytokine responses. We suggest that the similar kinetics of T-cell recovery in the two patient groups may be explained by the susceptibility to apoptosis of the activated CD4(+) T-cells in the autografts of the HM patients. This susceptibility to apoptosis would interfere with a swift and sustained CD4(+) T-cell regeneration post SCT.

Regression of advanced ovarian carcinoma by intraperitoneal treatment with autologous T lymphocytes retargeted by a bispecific monoclonal antibody.

BACKGROUND: The high frequency of relapse after induction chemotherapy of advanced ovarian carcinoma calls for new therapeutic approaches. Lysis of ovarian carcinoma cells can be achieved by retargeting of T lymphocytes using F(ab')2 fragments of the bispecific monoclonal antibody (MAb) OC/TR, which is directed to the CD3 molecule on T lymphocytes and to the folate receptor on ovarian carcinoma cells. PURPOSE: Our purpose was to assess in ovarian carcinoma patients the antitumor activity of in vitro-activated autologous peripheral blood T lymphocytes retargeted with OC/TR. METHODS: Patients with epithelial ovarian cancer (International Federation of Gynecology and Obstetrics stages III and IV) meeting specific criteria were eligible to enter a phase II immunotherapy trial. Before immunotherapy, the 28 patients who entered the trial underwent laparotomy to reduce their tumor load and to allow measurement of all indicator lesions. They then received two cycles of five daily intraperitoneal infusions of autologous in vitro activated peripheral blood T lymphocytes retargeted with OC/TR plus recombinant interleukin 2 (IL-2) with (n = 11) or without (n = 17) a second daily infusion of OC/TR F(ab')2 and IL-2. Response to treatment could be assessed in 26 patients following explorative laparotomy; time to progression could be assessed in 27 patients. RESULTS: Seven patients had clinical evidence of progressive disease after treatment and therefore did not undergo laparotomy. Of the 19 patients evaluated by surgery and histology, three showed complete response, one showed complete intraperitoneal response with progressive disease in retroperitoneal lymph nodes, three showed partial response, seven had stable disease, and five had progressive disease. The overall intraperitoneal response rate was 27% (95% confidence interval [CI] = 10%-44%). The complete responses seen in three patients lasted 26 months in one patient, 23 months in the second, and 18 months in the third. Two patients were not assessable for response. One of these patients had bowel perforation during catheter removal, which precluded further evaluation. The second patient was positive only by cytologic examination before immunotherapy, was tumor free at laparotomy after immunotherapy, and remained so for the entire 21 months of follow-up, as determined by cytologic examination of random biopsy specimens. The median time to disease progression in the 15 assessable patients plus those who had stable disease was 11 months (95% CI = 6-18 months). Immunotherapy-related toxic effects included mild to moderate fever, nausea, emesis, and fatigue. Anti-mouse antibodies were detectable by the end of the treatment in 21 of 25 patients tested. CONCLUSIONS: Locoregional immunotherapy of ovarian cancer with bispecific MAb-retargeted T lymphocytes can result in tumor regression. Toxicity was mild to moderate and only transient. IMPLICATIONS: Improvement in systemic antitumor responses is needed before this approach can prove useful as adjunctive treatment following induction chemotherapy in patients with minimal residual disease.